Skin initiating action and lung carcinogenesis by derivatives of urethane (ethyl carbamate) and related compounds

Biochemical Pharmacdogy,

1959, Vol. 2, pp. 168-176. Pergamon Press Ltd., London

SKIN INITIATING ACTION AND LUNG CARCINOGENESIS BY DERIVATIVES OF URETHANE (ETHYL CARBAMATE) AND RELATED COMPOUNDS* I. BERENBLUM, D. BEN-ISHAI~, N. E. SIMON and Department

of Experimental

HARAN-GHERA,

A. LAPIDOT~,

N. TRAININ

Biology, The Isaac Wolfson Building, The Weizmann Science, Rehovoth, Israel

Institute

of

(Received 13 February 1959) Abstract-The possibility that a metabolite might be implicated in the carcinogenic action of urethane on the lungs, and in its limited “initiating” action in the case of skin carcinogenesis, prompted the testing of a number of derivatives and analogues of urethane for both lung carcinogenesis and skin initiation in mice. The modifications in molecular structure of urethane involved (i) changes in the carbamyl portion, (ii) changes in its carboethoxy portion, (iii) phosphorylated derivatives, and (iv) addition compounds with keto and amino acids. Most of the compounds tested proved inactive, and among those that did give positive results, the effects were weak; thus indicating that none of them represented the sought-for metabolite. Some tentative implications of the results are discussed as pointers for further enquiry.

in the biological properties of urethane and in its metabolic fate the animal body arose from the fact that, in addition to its carcinogenic action on the lungs2 and the fore-stomach3 in mice, and on the liver and lungs4 in rats, urethane also possesses a pronounced “initiating” action on mouse skin, either when applied locally5 f 6 or administered systemically,’ rendering the skin of such treated animals responsive to the “promoting” action of croton oi1.l The problem of the metabolism of urethane was further stimulated by the observation of Rogers*? s on the possible participation of (unidentified) metabolites in the neoplastic transformation of lung tissue by urethane. Attempts to determine the nature of the postulated metabolite of urethane (as distinct from the end-products, ethanol, ammonia and carbon dioxide, which are manifestly not implicated) by metabolic studies involving 14C-labelled urethane, have so far been unsuccessful.lO~ l1 The present investigation represents another, more empirical, approach, in which a series of urethane-related compounds that could conceivably be intermediate metabolites of the parent compound, were tested for skin initiation and for lung carcinogenesis in mice. If any one of these proved to be at least as potent as urethane itself, the possibility of it representing a key metabolite responsible for the action of the parent compound could serve as a pointer for further metabolic studies. The choice of compounds for testing was based not only on the known hydrolytic RENEWED interest in

* A preliminary report of these findings was included in a review by one of the authors (Berenblum)‘. t Present address Chemistry department, Haifa Institute of Technology, Israel. : Now working in the Isotope department, The Weizmann Institute of Science, Rehovoth, Israel.

Skin initiating action and lung carcinogenesis by derivatives of urethane

169

breakdown of urethane in the body, but also on possible oxidative mechanisms, as side reactions, and on addition compounds between body constituents and urethane itself or some of its postulated breakdown products. The compounds tested may conveniently be considered under four headings: (i) changes in the urethane molecule involving the carbamyl group; (ii) changes involving the carboethoxy group; (iii) phosphorylated derivatives; and (iv) addition compounds with keto or amino acids. METHODS

The animals used in these experiments were male and female mice of the Swiss strain, bred in these laboratories by brother-sister mating for from fifteen to twenty generations. They were 2-3 months old at the start of the experiment, kept in plastic cages, housed in an air-conditioned room at 21-23 “C, and fed on Purina Laboratory Chow and water ad Iibitum. The urethane derivatives and related compounds were synthesized by the following methods : The N-methylethylcarbamate, N : N-dimethylethylcarbamate, N-hydroxyethylcarbamate, allylcarbamate, carboethoxyglycine, carboethoxy-1-alanine, carboethoxyI-aspartic acid, and carboethoxy-I-glutamic acid were prepared from the corresponding amine or amino acid and ethylchlorocarbonate by the Schotten-Baumann procedure. The ~-hydroxyethyl~rbamate was prepared from ethylene glycol and ammonia; the 2-oxazohdone by the method of Homeyerra; and the @tminoethylcarbamate by the method of Ben-Ishai Is. The carbamyl phosphate was prepared as the lithium salt by the method of Jones et aAl and this was converted to the sodium salt by passing through an ion exchange column of Amberlite IR-400. The urethane phosphate was prepared by the method of Lapidot and HalmanrF’; while the carboethoxyphosphate, which had not previously been synthesized, was prepared by Hahnann and Lapidot by catalytic hydrogenation of dibenzylcarboethoxyphosphate. (It should be noted, however, that this substance proved too unstable for satisfactory characterization, and its use for biological testing might, therefore, have largely involved decomposition products.) The monothiourethane was prepared according to the method of Pinne9; the other sulphur analogue, xanthogenamide, according to that of Debusl’. The urethane was obtained from British Drug Houses Ltd., England; the propyl urethane, butyl amine, diethyl carbonate and ethylene carbonate from Eastman Kodak, Rochester, U.S.A.; the “Miltown” from Lederle Laboratories, New York, U.S.A.; and the croton oil from Magnus, Mabee & Reynard, New York, U.S.A. Each compound was administered once only, either by mouth or intrap~ritoneal injection, as aqueous solution in the case of the higher dose ranges, in saline in the case of lower dose ranges, and in medicinal liquid paraffin for compounds that were relatively insoluble in water. The original intention of using molar equivalents had to be abandoned in view of the varying toxicities among the compounds. After an interval of 4 days following the injection, the animals were submitted to twice-weekly applications to the skin of a 5 per cent solution of croton oil in liquid parafIin, for 40 weeks, the applications being limited to an area of about 2 x 2 cm in the dorsal region over the shoulder blades. The croton oil controls were treated the same way, except that they did not receive any preliminary intraperitoneal injection. The resulting ckin tumours were charted at their fI.rst appearance, and fortnightly

170

I.

BERENBLUM,D. BEN-ISHAI, N. HARAN-GHERA, A. LAPIDOT, E. SIMON and N. TRAININ

thereafter; but papillomas that regressed within 2 weeks of their first appearance were not listed in the final records. All the animals were killed at the end of the croton oil paintings, autopsied and the lungs kept for histological examination, to determine more accurately the number of adenomas present in that organ than was possible by visual examination. RESULTS

Table 1 summarizes genesis, as determined applications of croton

TABLE TONEAL

1.

the results, both for skin initiating action and lung carcinoat the end of the experiment, i.e. after completion of eighty oil (40 weeks). Since the number of skin tumours after such

SKIN INITIATING ACTION AND LUNG CARCINOGENESIS

INJECTION (i.p.)

OR ORAL

ADMINISTRATION

Results after 40 weeks (eighty applications)

(p.0.)

of subsequent

BY A SINGLE INTRAPERI-

OF URETHANE

Compound

DOS ,e

Ethyl carbamate (urethane) Ethyl carbamate Propyl cart&ate N-Methyl urethane N-Methyl urethane N:.WDimethyl urethane N-Hydroxy urethane N-Hydroxy urethane N-Hydroxy urethane N-Hydroxy urethane Ally1 urethane Ally1 urethane &Hydroxy urethane &Amino urethane-HCI Oxazolidonc Oxazolidone Monothiourethane Xanthogenamide Butylamide Diethyl carbonate Diethyl carbonate “Miltown” Carbamyl phosphate Carbamyl phosphate Potassium cyanate Carboethoxyphosphate Urethane phosphate Carboethoxyaspartic acid Carboethoxyglutamic acid Carboethoxyalanine Carboethoxyalanine Carboethoxyglycine

25

25 12.5 i 11.5 12.5 ;: ‘:‘” :‘2:: g 10 : 20 12.5 11.4 7 :: 2.5 : : 5 ;

T

Solvent

Route

aq. dist.

p.0.

16/18

i, nit i&ion _I_ -1Average no turnours pa r mouse -_ 1.6 * 0.3 89%

saline

i.p.

18/30

60%

rlumbet

aq.&. $$st. liq. par. aq. dist. aq. dist. saline aq. dist. aq. dist. saline liq. par. aq. dist. aq. dist. saline saline saline saline aq. dist. saline saline aq. dist. aq. dist. saline saline saline saline saline saline saline saline

p.0. p.0. i.p. p.0. p.0. I.P. i.p. i.p. p.0. 1.p. p.0. p.0. p.0. 1.p. i.p. i.p. i.p. p.0. 1.p. i.p. p.0. i.p. 1.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p.

Skin

._

Tumour bearing/ survivors

(mg)

‘I -_

’ :; 30 40 10

8/24

33%

:8

-

-

DERIVATIVES

croton oil treatment

0.9

*

0.2

0.4 0.3 1.2 0.4 0.4 1.5 1.9 0.2 0.25 0.8 0.25 0.4 0.25 0.9 0.35 0.45 0.15 0.55 1.1 0.4 0.3 0.85 0.55 0.6 0.4 0.4 0.55 0.3 “0:;

f 0.1 * 0.1 r 0.4 * 0.1 *0.1 + 0.3 + 0.2 f 0.1 f 0.05 f 0.2 zt 0.1 * 0.1 I 0.1 + 0.3 f 0.1 * 0.15 + 0.05 & 0.15 * 0.2 + 0.1 * 0.1 + 0.2 * 0.2 f 0.2 & 0.2 f 0.1 * 0.15 * 0.1 $ ;I;

0.3

*

Lung

carcinogenesis

Tumour bearing/ survivors

’ Average

no. turnours per mouse

l2/18

67%

3.4

23/26

88%

2.8

Z/42 4/14 4/20 7/24 O/l9 9/20 ll/lS 5/26 S/18 O/24 z/29 l/14 l/8

5% 0.05 29% 0.3 20% 0.25 29%,0.4

’

450/ 61% 19% 28% G 7 2 12%

% 1.3 + 0.5 i 0.03 & 0.1 + 0.1 ztO.1

0.75 10.3 +r 0.2 i 0.1 0.4 z 0.1

10.8 10.3

0.0711; 0.05 0.07 * 0.05 0.1 io.1

0.1 -

prolonged treatment with croton oil is relatively high in the controls, due to background carcinogenicity of the croton oil itself, the tumour yields in the various groups after only forty applications (20 weeks) are presented separately in Table 2. These data naturally refer to skin tumours only, since lung adenomas cannot be recognized during life. The background carcinogenicity of croton oil, at the twentieth week, being relatively low, makes it easier for the significance of differences between experimental and control groups, in the case of skin turnouts, to be assessed at that stage. Since the varying dosage prevents quantitative comparisons of intensities of action, the results are finally summarized in Tables 3-6 on a semiquantitative basis, i.e. by plus and

Skin initiating action and lung carcinogenesis

by derivatives of urethane

171

minus signs, indicating overall differences without claiming accuracy about minor differences, TABLE

2. SKIN INITIATING ACTION BY A SINGLE INTRAPERITONEAL INJECTION (i.p.) ORAL ADMINISTRATION (p.0.) Results after 20 weeks (forty applications)

OR

OF URETHANE DERIVATIVES of subsequent croton oil treatment

-

1.5 * 0.2 0.6 f 0.1 0.07 * 0.05

;:x:

0.2 & 0.1 0.3 & 0.08 0.17 i 0.08

25 10 -

aq. dist. saline -

Propyl carbamate N-Methyl urethane N-Methyl urethane N:N-Dimethyl urethane N-Hydroxy urethane N-Hydroxy urethane N-Hydroxy urethane N-Hydroxy urethane Ally1 urethane Ally1 urethane ,!I-Hydroxy urethane B-Aminourethane-HCl

25 12.5 11.5 125

aq. dist. aq. dist. saline 2. dys;

;: 11.8

aq: dist: saline aq. dist. aq. dist. saline liq. par. aq. dist. aq. dist. saline saline saline saline aq. dist. saline saline aq. dist. aq. dist. saline saline saline saline saline saline saline saline

Oxazolidone Monothiourethane Xanthogenamide Butylamide Diethyl carbonate Diethyl carbonate “Miltown” Carbamyl phosphate Carbamyl phosphate Potassium cyanate Carboethoxy phosphate Urethane phosphate Carboethoxyaspartic acid Carboethoxyglutamic acid Carboethoxyalanine Carboethoxyalanine Carboethoxyglycine

2; 125 11.4 7 2 2.5 : : 5 1 1

Average no. tumours per mouse

p.0. 1.P.

Ethyl carbamate urethane) Ethyl carbamate Iurethane) Croton oil (control)

;: 25 10 5

Tumour bearing/ survivors

Route

bg

1:.5 12.7

No.

Solvent

-

i.p. ;::: i .p. &p. I$. lz. ;::: i.p. i.p. i.p. ;.“d. i.p. ;.:. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p.

2124 3140

8% 7%

0.7 10. 0.1 0.3 0.2 0.1 0.2 0.1 0.05 8’;;

10.2 * 0.2 & 0.05 & 0.1 * 0.05 * 0.05 * 0.1 & 0.01 & 0.05 ; ;‘;;

0.12 0.3 0.2 0.13 0.05 0.1 ;‘;5

* + * & * i: +

0.07 0.1 0.1 0.05 0.05 0.01 8’71

0.15 0.12 0.08 0.08

2 * & +

0.1 0.05 0.05 0.05

0.1 To.05 0.05 f 0.05

DISCUSSION

It was hoped that one or other of the tested derivatives would prove more potent than urethane itself and thus provide a lead as to the nature of the postulated effective metabolite. This hope was not realized. On the contrary, the few compounds that did elicit a positive response, whether for skin initiation or lung carcinogenesis, were decidedly weaker in action than urethane. The question then arose whether the complete lack of activity of some of the compounds might throw light on the problem of urethane metabolism in a negative sense, i.e. by enabling one to eliminate certain metabolic mechanisms that might otherwise have been considered possible on a priori grounds. On the assumption that the effective metabolite is an intermediate product in the hydrolytic breakdown of urethane, one might suspect either the carbamyl or the carboethoxy group to be the essential portion of the urethane molecule involved in

172

I. BERENBLUM,

D. BEN&W, N. HARAN-GHERA, A. LAPIDOT,E. SIMONand N. TRAININ

the biological action, reacting either directly with constituents of the living cell, or indirectly in the form of carbamyl or carboethoxy derivatives. Carbamyl portion NH,-CO-0-C2H, / I, Y Carboethoxy portion Direct action of the free, unstable, radical could not be determined by the experimental methods discussed here; indirect action by derivatives could, however, be examined by testing the suspected complexes, synthetically produced, for skin initiating action and lung carcinogenesis. Two classes of such complexes were examined: (a) phosphorylated derivatives (Table 5); and (b) complexes of urethane with ketoacids, which may also be considered as carboethoxy derivatives of amino acids (Table 6). It was found, however, that none of the compounds in either series had more than borderline activity for skin initiation or lung carcinogenesis. Most of them were, in fact, completely inactive. TABLE 3. MODIFICATION OF THE CARBAMYLPORTION OF THE URETHANE MOLECULE Initiating action

Formula

Compound

-_ H \

Ethylcarbamate (urethane) !

N-Methylethylcarbamate

i H’ I H,C I \ /

0 I’

on skin

+-f--t++

N~--O--C~H~

Carcinogenic action on the lungs

-t++++

0

N-k-0-CpH6

++

H’ H,C \ bamate

0 I/ N-C-0---C2H5

-

/ 0

HO

‘\

N-Hydroxyethylcarbamate

/I N-C-0-C2H,

i

/ H H

S ‘\

Xanthogenamide

N&-O-&H,

/

H’

H Oxazolidone

Diethyicarbonate

\

/ I j

0 // X’-C-O---CH~~~H2

!

/ :

-t-f

Skininitiating action and lung carcinogenesis TABLE 4. MODIFICATION OF THEI CARBOETHOXY

by derivatives of urethane

173

PORTION OF THE URETHANE

MOLECULE

Compound H Ethylcarbamate

Carcinogenic action on the lungs

Formula

(urethane)

0 ‘N&H

+++

5

I-+

+++++

H’ H

0

!I

\ Propylcarbamate

N-C-CIH,

+

+

+

-

(?) +

-

H’ H

0 \

Allylcarbamate

N-C-CH,CH,=

CH,

H’ H

0 \

fi-Hydroxyethylcarbamate

il

N-C-C,H,-OH

H’ H

0 \

fl-Aminocthylcarbamate HCl

N-A-O-C,H,NH,.HCl

-

-

0 i

-

H’ H \

Monothiourethane

0 II N-C-S-C,H,

H' H Butylamide

\

N-C4H,

+

-

-

_

H’ 0

HzN-C-0-CH, 2-Methyl-2-n-propyl 1:3-propane diol dicarbamate (Miltown) HaN-C-O-CH, k

CH, ‘C’ /\

C3H7

The fact that, among the phosphorylated derivatives some borderline activity (initiating action on the skin) was noted in the case of carbamyl phosphate, was surprising, since this compound is known to be a normal constituent of the body. One possible explanation of the result-that the preparation might have contained some urethane as contaminant-was rigorously excluded by chemical tests. The original

174

I. BERENBLUM, D. BEN-ISHAI,N. HARAN-GHERA, A. LAPIDOT,E. SIMONand N. TRAININ

experiment was then repeated, and slight initiating action noted once again (see Table 5). These results should, nevertheless, be accepted with reservations, being based on narrow differences between the experimental and control values. ~e~w~le, the search was carried a stage further, on the basis of the observatior+ that carbamyl phosphate liberates cyanate on decomposition under certain conditions. The latter was, therefore, also tested for skin initiation and lung carcinogenesis, but found to be inactive. Carboethoxyphosphate also appeared to be active, at least for lung carcinogenesis, though the results were not decisive in view of the poor survival of the treated animals due to the toxicity of the material. TABLE 5. PHOSPHORYLATEDDERIVATIVES

Compound

Initiating action on Sk&l

Formula H

_/-

Carcinogenic action on the 1UXlgS

0

NA---o--C;H, +-k-i-++ / H \

Ethylcarbamate (urethane)

HO Urethane phosphate

+-t--t.++

HO

m rt:

O>-&-C&II

(3

-c

HO 0

I-IO

-

i

(9 f

-

0+0-&-O-C& HO H

0

Carbamyl phosphate H’ Potassium cyanate*

I

KCNO

‘OH (3

rir

-

* Decomposition product of carbamyl phosphate.

Another approach was to test “artificial” derivatives of urethane (i.e. those not suspected of appearing metabolically in the body), with substituents in the carbamyl and in the carboethoxy portions, respectively, of the urethane molecule. If such derivatives invariably failed to show activity when the substituent was at one end of the molecule, but retained activity when the substituent was at the other end, the argument could be considered that the group in which “non-biological” substitution caused repression was the one essential for biological activity. From this point of view, the results (see Tables 3 and 4) were not conclusive, since considerable repression occurred in both series. But the fact that diethyl carbonate had any activity at all suggests that the presence of the amino (i.e. the carbamyl) group is not essential, and therefore, by implication, that the carboethoxy group might be the important group.

Skin initiating action and lung carcinogenesis by derivatives of urethane TABLE

175

6. COMPLEXES OF KETOACIDS WITH URETHANE, REPRESENTING AMINO ACID DERIVATIVES OF THE CARBOETHOXY

-

Initiating action on

Formula

Compound

GROUP

-

.-

_E~yl~b~ate (urethane)

Skill

+-t-+-l-+

carcinogenic action on the 1Ungs

-l-+-l-++

Carpdethoxyaspartic

H CATthox

0 II

\N

yglutamic

-8 / WOOC-CHC&,COOH

H6

H \

;7 +-C-+-GHs

H&--C~OOH H \

Carboethoxyglycine

? N--c-Q-GHs

C&NH -

-

A point of particular interest was that both sulphur analogues of urethane were virtually inactive. This is in keeping with the idea that the carboethoxy portion of the urethane molecule must remain intact for effective biological action. If the carbamyl were the critical portion, one would have expected only one of the two sulphur analogues to be inactive, namely the monot~oure~ane, i.e. with the sulphur substitution in the carbamyl portion. When the sulphur substitution is in the carbonyl group, both portions are naturally implicated. In a study of the carcinogenicity of a series of esters of carbamic acid, with respect to lung carcinogenesis, LarseP found that the high activity, in the case of the ethyl derivative, rapidly falls off when this group is replaced by isopropyl, and more so with the n-propyl, while the methyl, n-butyl and isoamyl derivatives are inactive. This indicated not only that the structural integrity of urethane is more critical for lung carcinogenesis than for its anaesthetic and other biological properties, but also suggested that the carboethoxy group is particularly important. On the other hand significant activity was also observed with tribromoethyl carbamate. In a subsequent investigation by Larsenzo, a similar study was made with ~-alkylated derivatives, i.e. with substitution at the carbamyl end of the urethane molecule. While activity was reduced in many cases, the specScity was not as critical as in the case of carbamic acid esters. Thus, fairly high activity was found in the case of isopropylurethane and methylene and ethylene diurethane, and lower activities with many of

176

I. BERENBLUM, D. BEN-TSHAI, N. HARAN-GHERA,A. LAPIDOT,E. SIMONand N.TRAININ

the other derivatives. These results add further weight to the belief that carcinogenic activity is dependent on the carboethoxy rather than the carbamyl group in the urethane molecule being intact. Reference should also be made to a recent report 21 that isopropyl-N-phenyl carbamate and isopropyl-N-(3-chlorophenyl) carbamate are weak but definite initiators for mouse skin carcinogen&s. Finally, there is the possibility that the effective metabolite might arise not as an intermediate product of the ~y~r~~yt~c breakdown of urethane, but by some oxidatke mechanism, through a side reaction. Though such a metabolic mechanism has never been demonstrated in animals, some evidence for it was suggested by Isenberg et a1.22, though subsequent work by that group cast doubts on their earlier interprctations.2” It should be noted, in this connexion, that in our investigations, N-hydroxyurethane had a decided action, both as skin initiator and lung carcinogen, though with a lower potency than that of urethane. However, hydroxyethyl urethane was inactive. (In the preliminary report,l the latter compound was also listed as a weak initiator, but more complete analysis convinced us that the observed differences between it and the control were not statistically significant.) While these observations may serve as pointers for further study, they do not permit any overall conclusion on the essential problem that prompted the present investigation. REFERENCES I. 1. BERENBLUM, Ann. R. COIL Surg. Engl. 21, 239 (1957). 2. A. NETTLESHIP and P. S. HENSWAW,J. Nut. Cancer Inst. 4, 309 (1943). 3.1. BERENBLUM and N. HARAN-GHERA,Cancer Res. 17, 329 (1957). 4. W. G. JAF& Cancer Res. 7, 107 (1947). 5. A. GRAFFI,E. VL~~YNCH, F. HOFFMANNand I. SCHULZ,Arch. Gesc~3v~ls~#r~e~.5, 110 (1953). 6. M. H. SALAMANand F. J. C. ROE, Brit. J. Cancer 7,472 (1953). 7. N. HARAN and I. BERENBLUM, Brit. J. Cancer 10, 57 (1956). 8. S. ROGERS, J. Nat. Cancer Inst. 15, 1675 (1955). 9. S. ROGERS,J. Exp. Med. 105, 279 (1957). IO. H. E. SKIPPER,L. L. BENNE~, JR., C. E. BRYAN, L. WHITE,JR., M. A. NEWTONand L. S~PSON, Cancer Res. It,46 (1951). I I. I. BE~NBLUM, N. HARAN-GHERA,R. WINNICKand T. WINNICK, Cancer Res. 18, 181 1’1958). 12.A. H. HOMIEYER, U.S. Pat. 2399118; Chem. Abstr. 40,4084 (1946). 13. D. BEN-ISHAI,J. Amer. Chem. Sot. 78, 4962 (1956). 14. M. E. JONES,L. SPECTOR and F. LIPMANN.J. Amer. Chem. Sot. 77, 8 19 (1955). 15. A. L.APIDOT and M. HALMANN,J. Chem. Sot. 347, 1713 (1958). 16. A. PINNER,Chem. Ber. 14, 1083 (1881). 17. H. B. DEBUS,Lie&gs Anna.75, 127 (1850). 18. M. E. JONES,L. SPECTORand F. LIPMANN,Proc. Third Int. Congr. ~i~c~e~r., Brussejir p. 278 (1955). 19. C. D. LARSEN,J. Naf. Cancer Insf. 8, 99 0947). 20. C. D. LARSEN,J. Nat. Cancer Insf. 9, 35 (1948). 21. G. J. VAN ESCH, H. VAN GENDEREN and H. H. VINK, Brit. J. Cancer 12, 355 (1958). 22. H. D. ISENBERG, A. SCHATZ, A. A. ANGRIST,V. SCHATZand G. S. TRELAWNY,J. Bacterial. 68, 5 (1954). 23. A. SCHATZ, G. S. TRELAWNY,V. SCHATZand R. R. MOHAN, Biochem. Bi0phy.r. Acta 21, 391

(1956).