15.9. 1976 Speeialia 1135

fers wi th the reduct ion of 3,5-dinitrosalicylic acid, the chromogen by reducing sugars, p roduc t of amylolysis ; and hence remains inconclusive. The size of the h y d r a t e d cat ion seems to be i r re levant , since CsC12 could ac t iva te the enzyme a lmost to the same degree as by sodium, po- t a s s ium and a m m o n i u m chlorides. Sodium bromide ac- t i va t ed the enzyme to some exten t , while b o t h NaNOa and CHaCOONa had no such action. The enzymic ac t iv i ty was inhib i ted by Na2COa as expected , since the p H of the react ion mix tu re was raised. These observa t ions regarding bromide and aceta te are also in ag reemen t wi th the earlier f indings. According to BERN~'ELD*, NO a- exhibi ts some ac t iva t ion b u t we failed to get the same as MYR- BACK and others.

In a recen t repor t 5, pe rhaps the only one so far, sodium ion has been claimed to be the ac t iva to r of pancrea t ic c~- amylases of toad, reptile, pigeon and rat. Using NaC1, Na~COa, KC1 and CuCI~ in p h o s p h a t e buffer, t h e y had shown t h a t while NaC1 and Na~COa ac t iva ted the enzyme b o t h CuCI~ and BIC1 did not . I t is a l ready repor ted in the l i te ra ture s t h a t ra t sa l ivary and liver amylases are ac- t i va t ed by KC1, as also conf i rmed by us. The failure to get any ac t iva t ion by CuCle is r a the r to be expec ted and the reasons are discussed earlier. Unlike the f indings of these

authors , ac t iva t ion by Na2CO a could no t be conf i rmed in our l abora to ry since addi t ions of Na2CO 3, as low as 2.5 / ,moles in the 2 ml of react ion mix tu re caused a change in pH. However , wi th Tris-carbonate buffer a t p H 7.0 similar ac t iv i ty was ob ta ined as wi th Tris-phosphate. I t can be men t ioned here t h a t me thodo logy ad ap t ed and concen t ra t ions of KC1, CuCI~ and Na~CO3 used by us were the same as repor ted by themS.

In view of the above, the following conclusions have been drawn. Chloride ion shows increased ac t iva t ion from p H 6.0 to 7.0 while mos t o ther anions including phospha t e do the reverse. Act iva t ion by chloride ion is h igher com- pa red to o the r ions a t bo th the p H values. The presence and size of the cat ion is i r relevant . Essen t i a l i ty of Ca++ as an t i c ipa ted t h ro u g h earlier inves t iga t ion can ev iden t ly be ruled out as shown by the resul ts of oxalate , ace ta te and a spa r t a t e in Tris buffer.

6 p. BERNFELD, Methods in Enzymology (Academic Press, New York 1955), vol. 1, p. 149.

7 S. K. MEUR, V. S. RAO and K. B. DE, Proc. Indian Sci. Congr. (1975), part 3, p. 69.

8 R. L. McGEAcHIN and B. A. POTTER, J. biol. Chem. 235, 1354 (1960).

The Oxidat ive M e t a b o l i s m of t z -Chlorohydr in and the C h e m i c a l I n d u c t i o n of S p e r m a t o c o e l e s

A. IR. JONES 1 and Carole MURCOTT

Pharmacology Department, University of Manchester, Manchester M13 9PL (England), 16 February 1976.

Summary. a-Chlorohydrin (I) is ox ida t ive ly metabol ized to /3-chlorolactic acid (III) and oxalic acid (IV). Deposi t ion of calcium oxalate wi th in the renal tubules is responsible for t he toxic effects of c~-chlorohydrin and a similar act ion on the epid idymis or ep id idymal blood vessels could ini t ia te the format ion of spermatocoeles f rom this and o ther male an t i fe r t i l i ty agents.

The male ant i fer t i l i ty agen t 2 c~-chlorohydrin (3-chloro- propan- l ,2 -d io l , I) has two effects on the male ra t re- p roduc t ive t ract . Consecut ive low dai ly doses (5 • 10 mg/ kg) cause an immedia te and reversible phase of infer- t i l i ty 3 by inhibi t ing spe rm glycolysis 4, whereas a single high dose (100 mg/kg) produces ep id idymal lesions or spermatocoeles 5. These lesions occlude the ductul i ef- ferentes, block the passage of tes t icu lar spe rm and pro- duce prolonged or even p e r m a n e n t infert i l i ty. As the main metabol i te 6 of r the epoxide glycidol (2 ,3-epoxypropan- l -o l , II) has a similar ant i fer t i l i ty act ion to the low dose regime of e -ch lorohydr in 3, bu t does no t induce spermatocoele format ion, the possibi l i ty arose t h a t ~-chlorohydrin m a y be conver ted b y ano the r route to a metabol i te responsible for the ep id idymal lesions.

Chromatograms of ra t urine collected wi th in 8 h of e i ther oral or i.p. admin i s t r a t ion of a~Cl-c~-chlorohydrin (100 mg/kg) revealed the presence of 3 radioact ive com- ponents . These were ident i f ied as CI-, unchanged c~-

1 Present address: Biochemistry Department, University of Sydney, N.S.W. 2006, Australia. J. A. COPPOLA, Life Sci. 8, 43 (1969).

3 H. JACKSON, I. S. C. CAMPBELL and A. R. JONES, Nature, Loud. 226, 86 (1970).

4 H. MOHR�92 D. A. I. SUTER, P. D. C. BROWN-WOODMAN, I. G. WHITE and D. D. RIDLEY, Nature, Loud. 255, 75 (1975).

5 E. R. A. COOPER, A. R, JONES and H. JACKSOI% J. Reprod. Fertil. 38, 379 (1974).

s A. R. JONES, Xenobiotica 5, 155 (1975).

CHOH

I I CHzOH CH2OH

(I) (1)

CH2C[ i CHOH ~ COOH I } COOH COOH

~$) (~)

The metabolism of c~-chlorohydrin in the Rat. fl-chlorolactic acid (BCLA, III) was isolated from the acidified urine of male Wistar rats dosed with cr (100 mg/kg), by continuous ether extraction and chromatography on a column of Merck silica gel G, elution with ethyl acetate: 40-60 ~ petroleum ether (2:1) giving white plates, m.p. and mixed ~s in.p. 78-79 ~ Characterization was by reverse isotope dilution, mass spectroscopy {M+ 125/127, base peak 79/81 (M-H-CQ)} and GLC (retention time of a spiked sample 11.30 m on a 1 m• inin i.d. 5% FFAP on 80-100 mesh Chromosorb (~ at 150 ~ with N 2 inlet pressure of 14 psi). Urinary 0r chlorohydrin (I) was identified as the bis-benzoyl derivative as previously described 6, chloride ion by the method of SEILER and KAF~ENBElaGER ~1 and oxalic acid (IV) according to PARKE and WILLIAMS 2~. asC1-BCLA and uniformy-labelled 14C-BCLA were pre- pared from a6Cl-~-ehlorohydrin~ and uniformly-Iabelled 14C-c~-chlore- hydrin e3, respectively, by mild nitric acid oxidation 2~. Rf values (Merck pre-coated silica gel G TLC plates, 0.25 mln) were 0r hydrin (0.73), BCLA (0.30), calcium oxalate (0.10) and CI- (0.01- 0.15) in chloroform : methanol (7 : 3).

1136 Specialia EXPERIENTIA 32/9

chlorohydr in and an acidic metabol i te , fl-chlorolactic acid (BCLA, I I I ) . During the 24 h after adminis t ra t ion of e-chlorohydrin, 16% of the radioact ive label is e l iminated as a~C1- indicat ing extensive dehalogenat ion by epoxi- dat ion (I->II) , since a~C1- is excreted slowly due to dilu- t ion wi th the n o r m a l C l - pool 7. BCLA appears as a ur inary metabol i te 3-5 h after adminis t ra t ion of e-chloro- hydr in and continues to be excreted, toge ther wi th traces of a6Cl-, 7 days later. The a m o u n t of BCLA excreted in- dicates t h a t the oxidat ive metabol ic p a t h w a y ( I -+II I ) occurs wi th approx imate ly 25% of the e-chlorohydrin given a t this dose level. Adminis t ra t ion of BCLA (100 mg/ kg, oral or i.p.) to male rats, however, did not induce visible ep id idymal lesions though histological sectioning of the tes t is-epididymis complex showed the ductul i ef- ferentes to be ei ther di lated or choked wi th sperm, similar to the ear ly symptoms of spermatocoele format ion by e-chlorohydrin 5.

Dur ing these studies i t was observed tha t e-chloro- hydr in and BCLA (100 mg/kg) have a diuret ic act ion on male rats. Higher doses of ei ther compound (120-150 mg/ kg) cause anuresis and dea th by renal failure, his tology reveal ing glomerular nephri t is due to a crystal l ine renal deposition. Fur thermore , the urines of the diuret ic animals were examined microscopical ly and found to conta in envelope-shaped crystals, character ized as calcium oxal- ate. Consequent ly the metabol i sm of a6C1- and 14C-BCLA (100 mg/kg, i.p.) was inves t iga ted; whereas a6C1-BCLA appeared unchanged together wi th anC1- in the urine, r ad ioac t iv i ty f rom t~C-BCLA was localized in the kidneys and oxalic acid (IV), as calcium oxalate, identif ied as a ur inary metabol i te . Adminis t ra t ion of oxalic acid (50 mg/ kg, i.p.) to male rats caused a brief phase of diuresis and at au topsy approx ima te ly 50% of the t rea ted animals exhib i ted visible mono- la tera l spermatocoeles. Higher doses of oxalic acid were toxic due to renal deposi t ion of calcium oxalate 8 similar to the effects seen with high doses of bo th ~-chlorohydrin and BCLA.

The mechanism by which oxalic acid induces the forma- t ion of spermatocoeles is not known. I t m a y involve inter- ference wi th the reabsorpt ion of tes t icular fluid f rom the ductul i efferentes and caput epididymis to the blood s t ream 9 since in p r imary hyperoxalur ia , ex t ra rena l de- posits of calcium oxalate are f requent ly found in the walls of veins, arteries and arterioles associated wi th the male ur inogeni ta l t r ac t ~~ Previous studies have con- cluded t h a t spermatocoele format ion by a single high dose of ~-chlorohydrin involves interference wi th an ab- sorpt ive role of the epididymis 11 or conus epididymis 5

possibly by causing chemical changes in vascular per- meabi l i ty in this region 12,1a. If this act ion of e-chloro- hydr in is due to the sustained in v ivo release of oxalic acid, o ther compounds metabol ized to oxalic acid m a y also be expected to induce similar lesions. Long- te rm adminis t ra t ion of e thylene glycol mono-e thy l ether, which produces oxalic acid via e thylene glycolX4, is reported as causing bi lateral tes t icular lesions in over 60% of t rea ted male rats ~. Three other male ant i fer t i l i ty agents produc- ing spermatocoeles are e -bromohydr in 5. 1-amino-3-chlo- ropropan-2-ol*6 and e thane- l ,2 -d imethanesu lphona te ,7. I t is possible t ha t these three compounds could be me- tabolized to oxalic acid v ia fl-bromolactic acid, BCLA18 and e thylene glycol, respect ively. Pre l iminary results show t h a t these three ant i fer t i l i ty agents induce diuresis in male rats and t h a t the urine contains calcium oxalate. Their metabol i sm together wi th the effect of oxalic acid on the male ra t reproduc t ive t ract , is at present being invest igated.

7 L. G. WELT, in The Pharmacological Basis o/ Therapeutics, 4th edn. (Eds. L. S. GOODMAN and A. GILMAN; MacMillan, London 1970), p. 773.

8 M. G. MULINOS, L. POMERANTZ and M. E. LOJKIN, Am. J. Pharm. 715, 51 (1943).

9 B. CRABO, Acta vet. scand. 6, supll. 5 (1965}. 10 H. E. WILLIAMS and L. H. SMITH, in The Metabolic Basis o]

Inherited Disease, 3rd edn. (Eds. J. B. STANBURY, J. B. WYN- GAARDEN and D. S. FREDRICKSOH; McGraw Hill, New Y o r k 1972), p. 196.

1i S. A. GUNN, T. C. GOULD and W. A. D. ANDERSON, Proc. Sot. exp. Biol. Med. 132, 656 (1969).

12 R. J. ERICSSON, J. Reprod. FertiI. 22, 213 (1970). la E. SAMOJLIK and M. C. CHANG, Biol. Reprod. 2, 299 (1970). 14 p . .K . GESSNER, D. V. PARKE and R. T. WILLIAMS, Biocheln. J .

79, 482 (1961). 15 H. J. MORRIS, A. A. NELSON and H. O. CALVERY, J. Pharniac.

exp. Ther. 74, 266 (1942/. 16 j . A. Coppot~A and R. J. SALDARINI, Contraception 9, 459 (1974). IT E. R. A. COOPER and H. JACKSON, J. Reprod. Fertil. 34, 445

(1973). 18 Preliminary observations with a6CI-l-amino-3-chloropropan-2-ol

indicate that ~-chlorohydrin and BCLA are urinary metabolites of this antifertility agent1%

19 C, MURCOTT, M. Sc, Thesis, Manchester , 1976. 28 E. BAER, Biochem. Prepar. 2, 25 (1952). a H. SEILER and T. ]5~AFFENBERGER, Helv. ehim. Acta 44, 1282

(1961). 22 D. V. PARKE and R. T. WILLIAMS, Biochem. J. 5,l, 231 (1953). ~a A. R. JONES, J. Labell . Cpds. 9, 697 (1973). 24 C. F. KOELSCH, J. Am. chem. Soc. 52, 1105 (1930).

Developmental Characteristics of Histamine Methyltransferase and Phenylethanolamine -N-Methy l - transferase of Rat Brain

]~. D. I~OUVELAS 1, Ch. E. SAVAKIS, E. TH. TZEBELIKOS, G. BONATSOS and S. MITROSSILIS

Department o/ Physiology, University o/ Athens, Medical School, Goudi, Athens 609 (Greece), 79 February 1976.

Summary. The specific ac t iv i ty of h i s tamine methyl t ransferase of ra t brain increases rapidly f rom the 16th unti l the 25th d a y of gestat ion (7 days after birth). The specific ac t iv i ty of phenyle thanolamine-N-methyl - t ransfe rase shows a rapid increase dur ing the 1st and the 2nd week after bir th, the adul t values being obta ined by the end of the 2nd week.

His tamine migh t be a centra l neuro t ransmi t t e r 2 and a regula tor of tissue growth as well a. The ma jo r p a t h w a y for h is tamine metabo l i sm in the brain of cat, mouse and ra t is me thy la t ion of the imidazole ring to yieId 1-methyl ( -aminoethyl)- imidazole (methyl-his tamine) 4. His tamine

methyl t ransferase (HMT) is d is t r ibuted all over the brain of the rat, the highest levels being found in the hypo- tha lamus 5, ~.

Phenyle thanoDimine-N-methyl t ransferase (PNMT) in mammals is h ighly localized wi th in the adrenal medul la ~.