338 BIOCHIMICA ET BIOPItYSICA ACTA

BBA 96849

CHLORAL H Y D R A T E I N H I B I T S P R O T E I N SYNTHESIS IN VIVO

D A N I E L M c M A H O N AND W I L L I A M B L A S C H K O

Division of Biology, Calilornia Institute of Technology, Pasadena, Call[. 9iro9 (U.S.A .)

(Received N o v e m b e r io th , 197 o)

SUMMARY

A wide variety of substances can produce the unique inhibition of cell division characterized as c-mitosis and can also induce anesthesia. One of these is chloral hydrate. I t is more active, on the basis of its physical properties, than other compounds with the same effects. We investigated the basis of its unique effectiveness for the inhibition of cell division using the protozoan, Chlamydomonas reinhardii. At a con- centration which inhibits cell division, it completely inhibits protein synthesis in vivo, but has only slight effects on a number of other cellular functions. This inhibition is sufficient to explain its inhibition of cell division. In addition it is possible that this inhibition of protein synthesis by chloral hydrate may be relevant to its effects on the central nervous system.

INTRODUCTION

Chloral hydrate has two important biological effects; it effectively inhibits cell division in both plants and animals 1-~ and is an anesthetic for animals, from flat- worms to man 4. I ts effects on mitosis have been studied in two systems. In grass- hopper spermatocytes, it prevents the elongation of the mitotic spindle fibers but allows the contraction of chromosomal spindle fibers z. I t prevents the normal forma- tion of mitotic poles in dividing the endosperm tissue. Instead of two poles, three or more poles are formed or the chromosomes may show no polarity 1.

I ts quality of action as an anesthetic is ill defined. Observations that it slightly reduces oxygen consumption by the brain 5, have led to investigations which have shown that it can inhibit the respiration of brain slices in vitro with coordinate chan- ges in the concentrations of some metabolites 6. I t also inhibits electron transport in plant mitochondria 7. These effects occurred in much higher concentrations than those effective in vivo. Superficially its action as an anesthetic appears to be unrelated to its effects in cell division; however, circumstantial evidence argues for a link between the two processes. A number of compounds can promote anesthesia and prevent mi- tosis and they show a correlation between some of their physical properties and their minimum effective concentrations for both processes 3. Since some of these are not very reactive chemically (xenon, etc.), a number of physical mechanisms through which they might exert their effects have been proposed 8-12. The way in which these modifications might affect the biochemistry of the cell has been largely neglected. Chloral hydrate is more effective as an anesthetic and an inhibitor of cell division 3

Biochim. Biophys. Aeta, 238 (1917) 338-342

CHLORAL HYDRATE INHIBITS PROTEIN SYNTHESIS 339

than might be expected on the basis of its physical properties. Although one might guess that its effects on the cell would not be specific because of its chemical reactivity, at low concentrations it appears to be a specific inhibitor of protein synthesis in vivo.

MATERIALS AND METHODS

Chlamydomonas reinhardii, mt ÷, Strains 89 and arg 2, an arginine-requiring auxotroph, have been used. They were grown in high salt minimal medium l~ plus 24 mM sodium acetate. Except where noted, this medium was supplemented with 5-7" lO-4 M arginine for arg 2. Stock cultures were grown at 22 ° and a light intensity of lO 3 lux and were used during the exponential phase of growth. Chloral hydrate from Eas tman Kodak Co. was used either unpurified or it was purified by distillation in a Nester Rotating Band distillation apparatus, with no difference in the results. I t was used at a concentration of io mM, except where noted. Arginine and cyclohexi- mide were the ploducts of Sigma Chemical Co. 8H-labeled arginine and adenine were obtained from New England Nuclear Co. or from Amersham-Searle.

RESULTS AND DISCUSSION

Fig. I shows that chloral hydrate also inhibits cell division in C. reinhardii. Its effects range from a transient inhibition at 2.5 mM through a continued, but par- tial, inhibition at 5 mM and are complete at io mM. A concentration of io mM also very effectively inhibits protein synthesis. Fig. 2 illustrates an experiment where

o/ of the incorporation of [SH]arginine into protein. Because this ap- it inhibited 94 Jo parent inhibition of protein synthesis might be the result of trivial effects, such as an inhibition of the uptake of arginine into the cell, we have measured net protein synthesis by the method of LOWRY et al. z4 in Strain 89, a strain which requires no exogenous amino acids. No detectable increase in cellular protein occurs in chloral hydrate (Fig. 3)- We will show elsewhere that chloral hydrate prevents the light- stimulated induction of an enzyme, glutamate dehydrogenase 15. These expeliments indicate that chloral hydrate effectively inhibits protein synthesis in vivo. If this in- hibition is responsible for its inhibition of cell division, another inhibitor of protein synthesis should prevent cell division with similar kinetics. This is true. Cycloheximide (3.65 • lO -5 M) and chloral hydrate (IO mM) both begin to inhibit cell division after I h and the inhibition is complete after 4 h.

The experiments above do not exclude the possibility that the inhibition of protein synthesis is but one facet of a general inhibition of cellular functions. Since it has been shown to interfere with an oxidative electron transport in vitro (even though at high concentrations), this possibility was seriously considered. Two kinds of evidence make this unlikely. First the majori ty of the cells remained motile for over 24 h in chloral hydrate so, at a minimum, it does not seriously inhibit the synthesis of ATP or cause extensive changes in cellular permeability. Either of these effects, and others, would cause a loss of motility. This conclusion is reinforced by the data in Fig. 4. Chloral hydrate slightly increases the lag in the incorporation of adenine

Biochim. Biophys. Acta, 238 (i97 I) 338-342

340 o . MCMAHON, W. BLASCHKO

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Fig. I. Inhibi t ion of cell division by chloral hydrate . Cells of arg 2 were added to growth medium +arg in ine containing o ( 0 ) , 2.5 (O) , 5 ( ~ ) , io ( I ) , or 20 ( × ) mM chloral hydra te and samples were taken at intervals and counted in a Coulter Counter, Model A.

Fig. 2. Inhibi t ion of the incorporat ion of arginine into protein. Cells of arg 2 were added to growth medium + [SH]arginine (5 J*g and o.25 #C/ml). After 1.5-h al iquots of this culture were t ransferred to other flasks containing sufficient chloral hydra te to give final concentrat ions of o (O) , io (O) or 20 (A) mlVL During the course of the experiment , the cells increased 2.2-fold in the control f rom an initial concentrat ion of i . i • lO s cells/ml bu t showed no increase in number in either sam- ple containing chloral hydrate . At intervals i -ml aliquots were added to an equal volume of cold IO % trichloroacetic acid. After at least 0. 5 h at o ° they were heated to 9 °0 for 3 ° min and chilled to o ° again. They were washed onto W h a t m a n glass fiber filters (GF/A) wi th 3 × io ml of io % trichloroacetic acid and then washed successively wi th IO ml of 7 ° % ethanol (o°); twice wi th 7 ° % ethanol; twice with e thanol -e ther (I : I, v/v) and once with ether, all a t room tempera ture . The insoluble residue was digested in 0. 5 ml of hyamine hydroxide for 0. 5 h at 7 °o and after cooling io ml of toluene base scintillation fluid was added and the samples were counted in a Beckman scintillation counter.

into RNA, but its rate of incorporation in the presence of chloral hydrate rapidly equals or exceeds that of untreated cells. This suggests that there is no serious dis- ruption of RNA synthesis or of the production of the ribonucleotides needed for RNA synthesis. The incorporation of adenine into DNA is inhibited by 5 ° %. A similar in- hibition can be produced by other treatments which inhibit protein synthesis (star- vation for arginine; cycloheximide treatment; or in mutants conditionally defective in protein synthesis under nonpermissive conditions) and it is probably a secondary effect resulting from the inhibition of protein synthesis.

It is reasonable to assume that chloral hydrate exerts its effect either directly on a component of the protein synthesizing system, or through the alteration in the cell of a concentration of some molecule involved in protein synthesis or its control. In C. reinhardii the inhibition of cell division by IO mM chloral hydrate can be com- pletely explained by its effects on protein synthesis. We will show in another paper that there is also an excellent correlation between the ability of other concentrations to inhibit protein synthesis and their ability to inhibit cell division.

Protein synthesis is partially inhibited in liver by another anesthetic, chloro- form 16, and ethylether partially inhibits protein synthesis in the brain 17, but neither

Biochim. Biophys. Acta, 238 (I97I) 338-342

CHLORAL HYDRATE INHIBITS PROTEIN SYNTHESIS 341

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Fig, 3. Inh ib i t i on of ne t p ro te in syn thes i s by chloral hyd ra t e . Cells of s t r a in 89 a t a concen t ra - t ion of i .o • ioe cells/ml were i n c u b a t e d in g rowt h m e d i u m + o (O) or i o (O) mlV~ chloral h y d r a t e . At in te rva l s i -m l s amples were w i t h d r a w n a n d added to an equa l v o l u m e of io % t r ichloro- acet ic acid, kep t a t o ° for a t l eas t I5 m i n and t h e n cen t r i fuged a t 12 ooo × g for i min. This was r epea t ed wi th 5 % a n d t h e n io % t r ichloroacet ic acid. T h e y were washed wi th water , reeentr i - fuged, a n d al lowed to d ra in and were t h e n d iges ted in 2 ml of i M N a O H overn ight . After recen- t r i fuga t ion , p ro te in was m e a s u r e d b y t he LOWRY et al. 14 m e t h o d on an a l iquo t of t he s u p e r n a t a n t . L e a s t squa res regress ion ana lys i s of t h e d a t a ind ica tes t h e cont ro l ' s p ro te in c o n t e n t increased a t a r a t e of 3 . 4 / , g / m l per h while t h e cells in chloral h y d r a t e showed a ra te of increase of less t h a n 0 . 2 / , g / m l per h.

Fig. 4. The effects of chloral h y d r a t e on t h e incorpora t ion of aden ine in to nucleic acids. Arg 2 was a d d e d to g rowth m e d i u m con ta in ing o (O) or io (O) m M chloral h y d r a t e and [3H]adenine (0. 4/~C/ml, 22 C/mmole) . Samples were t a k e n a n d af te r a t l eas t 0. 5 h a t o ° t h e y were washed on to W h a t m a n GF/A fil ters wi th t h e p rocedure ou t l ined in t he legend of Fig. 2. Two samples a t each po in t were t h e n p repa red for coun t i ng to de t e rmine to ta l incorpora t ion in to R I ~ I A + D N A a n d two samples were i n c u b a t e d a t 37 ° for 4 ° m i n in 0. 5 IV[ N a O H . T h e n 0.6 ml of 5 o % t r ichloroacet ic acid was added to each vial and t h e y were kep t a t o ° for a t leas t 3 ° m i n and t h e n w a s h e d on to fi l ters once aga in and coun t ed b y t he p rocedures descr ibed in t h e legend to Fig. 2. These samples r ep resen ted incorpora t ion of aden ine in to DNA. All of t h e incorpora ted r ad ioac t iv i ty was m a d e soluble b y 0.5 h i ncuba t i on in 5 % t r ichloroacet ic acid a t 90 °, as is expec ted if i t is in nucleic acids. (a) Ef fec ts on incorpora t ion in to R N A wi th 6.o • lO s cells/ml. This incorpora t ion is largely in to h igh molecu la r we igh t RI~IA as ind ica ted by ana lys i s on sucrose g rad ien t s (M. MILLER, u n p u b l i s h e d resul ts) . (b) Ef fec t on incorpora t ion in to D N A in an e x p e r i m e n t wi th 6.9 • lO s cells/ml.

o f t h e s e i n h i b i t s p r o t e i n s y n t h e s i s t o t h e e x t e n t o f c h l o r a l h y d r a t e , a n d t h e p o s s i -

b i l i t y t h a t t h e e f f e c t s w e r e d u e t o a m o r e g e n e r a l d i s r u p t i o n o f c e l l u l a r m e t a b o l i s m

Biochim. Biophys. Acta, 238 (1971) 338-3 42

342 D. MCMAHON, W. BLASCHKO

was not excluded. These experiments show that an anesthetic can produce a major and relatively specific inhibition of protein synthesis in vivo. The possibility that chloral hydrate causes anesthesia by inhibiting protein synthesis or by inhibiting the production of a substance which is necessary for the function of both protein synthesis and the central nervous system is interesting. It is unlikely, however, that a simple general inhibition of protein synthesis could produce anesthesia. For cyclo- heximide, when injected into the brain, does not induce sleep but increases irrita- bility (G. MAGNUS, personal communication). The difference in the effect which cycloheximide and chloral hydrate produce on the central nervous system could be the result of different sites of action or different modes of action on the cell for the two compounds.

ACKNOWLEDGMENTS

W e t h a n k Mr. W i l l i a m F r y for his e x c e l l e n t t e c h n i c a l a s s i s t ance . Th is r e s e a r c h

w a s s u p p o r t e d b y U. S. Pub l i c H e a l t h g r a n t No. GMo6965

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Natl. Rech. Sci. Paris, (1954) 77. 4 H. M. KAPLAN, Federation Proc., 28 (1969) 1557. 5 J. H. QUASTEL, in J. LEvY AND P. GAVAUDAN, Mdcanisme de la Narcose, Coll. Intern. Centre

Natl. Rech. Sci. Paris, (1954) lO5. 6 H. MclLwAIN AND L. ]3UCHEL, Mdcanisme de la Narcose, Coll. Intern. Centre Natl. Rech.

Sci. Paris, (1954) 123. 7 W. S. PIERPOINT AND G. S. LATIES, Plant Physiol., 41 (1966) lO 5. 8 L. PAULING, Science, 134 11961) 15. 9 S. L. MILLER, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1515.

io J. A. CLEMENTS AND K. M. WILSON, Proc. Natl. Acad. Sci. U.S., 48 (1962) lOO8. i i H. H. MEYER, Arch. Exptl. Pathol. Pharmakol., 42 (1899) lO9. 12 K. W. MILLER, W. D. PATON AND E. ]3. SMITH, Brit. J. Anaesthesia, 39 (1967) 91o. 13 N. SUEOKA, K. S. CHIANG AND J. R. KATES, J. Mol. Biol., 25 (1967) 47. 14 O. H. LOWRY, N. J. ROSEBROOGH, A. L. FARR AND ]~. J. RANDALL, J. Biol. Chem., 193 (1951)

265. 15 C*. G6PEL, results to be published. 16 K. L. SCHOLLER, Experientia, 23 (I967) 652. 17 M. K. GAITONDE AND D. RICHTER, Proc. Roy. Soc. London, Set. B, 145 (1956) 83.

Biochim. Biophys. Acta, 238 (1971) 338-342