Similar effects of DDT and convulsive hydrazides on housefly metabolism

7. InsectPhysiol.,1966, Vol. 12, pp. 153 to 162. Pergamon Press Ltd.

Printed in Great Britain

SIMILAR EFFECTS OF DDT AND CONVULSIVE HYDRAZIDES ON HOUSEFLY METABOLISM RICHARD

E. CLINE

and GEORGE

W. PEARCE

Biology and Chemistry Section, Technology Branch, Communicable Disease Center, Public Health Service, U.S. Department of Health, Education, and Welfare, Savannah, Georgia, U.S.A. (Received 11 June 1965)

Abstract-Thiocarbohydrazide (TCH) was the most toxic of numerous convulsants tested in houseflies and was as toxic to a DDT-resistant strain as to a susceptible strain. The LD-50 values ranged from 0.3 pg/fly for injected aqueous TCH to 1 pg/fly for TCH dissolved in dimethylsulphoxide and benzene and applied topically. The in vko tracer studies with formateJ4C revealed that both DDT and TCH increased the labelling of purines while reducing the content of radioproline. Both toxicants stimulated the production of 14C0, from i4C-labelled proline and sugars, the increase from glucose-lJ4C being almost double that from glucose-6- 14C. Other similar effects of the toxicants involve the metabolism of l-carbon compounds in the presence of azaserine and urea production from arginine. Inverse metabolic effects exerted by DDT in the suceptible and resistant strains are discussed. INTRODUCTION

of the symptomatology of poisoning by DDT* in mammals (HAYES, 1959) and insects (PERRY, 1964) emphasize nervous system effects, the outward manifestations of which include hyperactivity, tremors, and convulsions. An increasing number of reports are concerned with the influence of insecticides on the levels of certain nitrogen compounds considered to be of particular importance to the nervous system. Selective proline depletion in the blood of roaches after poisoning with DDT was first described by CORRIGAN and KEARNS (1958). In roaches injected with 14C-proline DDT increased the level of radioglutamine in the blood and central nerve while decreasing the content of radioproline (CORRIGAN and KEARNS, 1963). In nerve cords of roaches DDT induced a fall in proline balanced by a rise in glutamine; and dieldrin, DFP, or o-IPMC caused a rise in DISCUSSIONS

* Amithiozone, 4-acetylaminobenzaldehyde-thiosemicarbazone; azaserine, o-diazoacetyl-L-serine ; DDT, 1 ,l,l -trichloro-2,2-bis(p-chlorophenyl)ethane; DFP, di-isopropyl 1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a phosphorofluoridate ; dieldrin, s5 96 ,7 ,8 98a-octahydroexo-1,4-endo-5, 8-dimethanonaphthalene; DON, 5-d&o-6-oxo-L-norleucine; o-IPMC, o-isopropoxyphenyl-N-methylcarbamate; metrazole, 1,5-pentamethylenetetraxole; TCH; thiocarbohydrazide; n-valone, 2n-Valery1 1,3-indandione. ?

153

154

RICHARDE. CLINE AND GEORGEW. PEARCE

ol-alanine as well as glutamine (RAY, 1964). The properties of an aromatic amine have been attributed to a neurotoxin which accumulates in the blood of DDTpoisoned roaches (HAWKINS and STERNBURG, 1964). In housefly experiments radiometric data indicated the accumulation of thoracic glutamine after treatment with DFP (WINTERINGHAM and HARRISON, 1956), and a similar effect was noted with DDT (WINTERINGHAM, 1958). In flies injected with 14C-formate, DDT and a number of other chlorinated hydrocarbon insecticides contrasted with other classes of insecticides tested in the induction of excessive radiopurine production along with radioproline depletion (CLINE and PEARCE, 1963). The severity of convulsions induced in rats was found to correlate with the brain concentration of DDT (DALE et al., 1963). Compounds found in abovenormal amounts in the brains of rats given a convulsive dose of dieldrin included carnitine and congeners (HOSEIN, 1963), ol-alanine and y-aminobutyric acid (WITTER and FARRIOR, 1964), and alanine, ammonia, lactic, and pyruvic acids (HATHWAY et al., 1965). In an effort to reveal new metabolic effects of DDT and compare its action with that of other convulsive agents present tracer studies were conducted in the housefly, Musca dowlestica. METHODS General procedure Adult female flies, maintained on water and sugar cubes for 4-7 days after emergence from the pupal cuticle, were treated topically on the abdomen with non-polar toxicants dissolved in benzene (1 ~1 of DDT solution/fly), or injected in the thorax with polar toxicants dissolved in water. Control flies received equivalent volumes of pure solvent. The non-toxicity of benzene has been discussed previously (CLINE and PEARCE, 1963). Food and water were withheld for the next 3 hr, after which each fly was injected at shallow depth in the side of the thorax, or in a few cases in the centre of the anterior part of the head, with l-2 ~1 of a 14Ccompound dissolved in water. Different metabolic effects have not been observed with the two different injection sites. In the case of 14C-formate the injection consisted of 1 ~1, 0.05 PC, 0.28 pg. The syringe used in injections had a 31-gauge needle at the tip. Injected control and treated flies in groups of ten were confined in 7 ml tissue grinder tubes (Corning Glass No. 7725)* for 3 hr, after which they were homogenized at room temperature, first in 2 ml of absolute ethanol, then in 2 ml of SOY& ethanol. Supernatants from the centrifugations of the homogenates were combined and used for assay or chromatography. Other experimental were as described previously (CLINE and PEARCE, 1963).

Radioactive Nuclear Corp.

compounds Azaserine

details

were purchased from Calbiochem and New England and DON were donated by Parke, Davis & Co.,

* Use of trade names and names of suppliers is for identification only, and does not constitute product endorsement by the Public Health Service.

SIMILAR EFFECTS OF DDT AND CONWLSIYB

dimethylsulphoxide ceutical Co.

by Crown Zellerbach

HYDBAZIDES

ON HOUSEFLY

METABOLISM

155

Corp., and metrazole by Knoll Pharma-

Radiorespirometry The tissue grinder tube with radioactive flies was fitted with a rubber stopper carrying an inlet tube reaching near the bottom and an outlet tube flush with the stopper. To trap 14C0, expired by the flies, air was drawn through the tube for 3 hr at a rate of 10 ml/min and bubbled through a mixture of 1 ml of ethanolamine and 2 ml of ethylene glycol monomethyl ether, a mixture described by JEFFAY and ALVAREZ(1961). The ten flies were then homogenized, first in 2 ml of absolute ethanol, then in 2.5 ml of 800/, ethanol, and the supernatants from centrifugations pooled and diluted to 5 ml with absolute ethanol. The residue from the alcoholic extractions was burned in a Coleman Carbon-Hydrogen Analyzer (Model 33), and the evolved r4C02 trapped in ethanolamine solution as above. To measure radioactivity a 0.2 ml aliquot of r4C02 trap solution or O-5 ml of the alcoholic extract was added to 20 ml of scintillation fluid and placed in a liquid scintillation counter (Nuclear-Chicago, Model 720). Th e scintillation fluid had the composition described by KINARD (1957) with toluene in place of xylene.

RESULTS Influence of DDT on the fate of injected 14C-compounds All the 14C-compounds listed in Tables 1 and 2 were injected into control and DDT-treated flies of two strains. Flies of the susceptible NAIDM strain received a lethal dose (0.4 pg/fly) of DDT whereas those of the resistant strain were treated with a much larger but non-toxic dose (10 pg/fly). In all the tracer experiments treated flies were compared with controls with regard to the production of soluble radiometabolites detected on paper chromatograms of extracts, and in many the output of 14C0, and the radioactivity of the insoluble residue were also measured. For experiments in which DDT had a marked influence on respiration the data are detailed in Table 1. Limited radio-respirometric data is included in Table 2 for cases where DDT had little effect on 14C02 output in either strain. It is to be noted in Table 1 that in the NAIDM strain DDT increased the 3 hr production of 14C0, from proline-5-r4C by 44 per cent and from proline-U-14C by 39 per cent. In the resistant strain, on the other hand, it can be seen that treatment with a much larger dose of DDT which does not elicit symptoms of poisoning reduced 14C0, formation from proline-5-14C by 23 per cent and from proline-U-14C by 23 per cent. In two other experiments with this strain decreases of 13 per cent and 20 per cent were obtained with proline-5-14C. The alcoholic extracts were shown by paper chromatography to contain largely proline and lesser amounts of glutamine and glutamic acid as radiometabolites. Interestingly, no metabolic effect was noted in radioproline-injected flies of a dieldrin-resistant strain after treatment with a large non-toxic dose of dieldrin (2 pg/fly).

156

RICHARD E. CLINE AND GEORGE W. PEARCE

The production of 14C0, from labelled sugars was increased in DDT-treated flies of the NAIDM strain but essentially unchanged in the other strain, as shown by the data of Table 1. Significantly the increase from glucose-1-14C can be seen to be almost double that from glucose-6J4C, suggesting a DDT-stimulation of the pentose phosphate pathway of metabolism by analogy with results reported by AGOSIN et al. (1963) for Triatoma infestans. Chromatograms did not reveal TABLE I-RECOVERY

OF RADIOACTIVITY(%/FLY) 3 HR AFTERINJECTIONWITH A %-COMPOUND NAIDM

Injected compound (pg, cpm/fly) n-Clucosamine-l-i%

Fractions

(5.4, 126,000) COa Extract Residue

strain

DDT-45 strain

Control

DDT

Control

DDT

30-6 37.1 22.8

46.1 26.4 18.1

17.4 47.8 31.3

20.2 50.8 23.5

rr-CIucose-l-14C (4.3, 57,000)*

COz Extract Residue

38.7 28.6 20.1

73.0 13.0 6.2

39.0 31.6 21.9

39.4 33.5 23.5

n-Glucose-6-i4C

CO, Extract Residue

33.4 34.2 24.1

47.9 26.6 15.3

34.5 37.1 25.9

39.8 33.3 24.9

(0.07, 38,000)

CO, Extract Residue

46.4 24.9 16.3

78.1 5.0 4.3

35.5 33.3 24.5

37.8 27.3 28.3

(22.8, 77,000)

CO, Extract Residue

49.0 36.0 4.9

70.7 15.3 7.7

58.7 25.4 6.2

44.9 39.0 4.8

co,

62.4 24.4 7-9

86.9 5.4 4.5

70.1 16.2 8.9

53.9 26.4 8.0

~-GIucose-U-~~C

nL-Proline-5J4C

(2.2, 99,000)*

L-Proline-U-i4C (0.035 , 105 ,000)

Injections were performed 1 ,ug/DDT-45 fly). * Injected into the head.

Extract Residue

3 hr after treatment with DDT

(0.4 pg/NAIDM

fly,

markedly different labelling patterns after injection of the different glucose By far the predominant radiometabolite of glucose in the extracts substrates. appears to be ol,ol’-trehalose, since it did not separate from the authentic trehalose on a silica gel-G-coated plate developed in n-butanol : pyridine : water (75 : 15 : 10) or isopropanol : acetic acid : water (90 : 30 : 30). A spray reagent containing anisaldehyde and sulphuric acid was used. The concentration of the trehalose was greatly reduced in NAIDM flies after treatment with DDT. In flies injected with the W-compounds listed in Table 2 changes due to DDT were noteworthy only in the NAIDM strain, and consisted of radioproline depletion whenever proline was a principal metabolite, increased labelling of

SIMILAREFFECTSOF DDT ANDCONWLSIVEHYDEAZIDESON HOUSEFLYMETAEOLISM 157

purines (uric acid and allantoin) from glycine-1-14C or glycine-2-14C, and overproduction of radiourea from citrullineJ4C or arginine-14C. Of the amino acids injected it is apparent that alanine is most readily utilized for energy production, and that hydroxyproline may not be an intermediate in the degradation of proline. TABLE ~-RADIOCARBON UPTAKEIN UNTREATEDNAIDM

Injected compound (pg,pc/fly)

L-Alanine-lJ4C (31, 0.1) y-Aminobutyric acid-l -ICC (21, 0.04) L-Arginine, guanidino-‘*C (0.56, 0.04) DL-Aspartic acid-3-14C (1, 0.05)

Uptake in 14C0 (%) a 81 32 1

n-Butyric acid, sodium-3-i4C (6.5, O-1) Caproic acid, sodium-l-14C (6.9, 0.05) L-Citrulline, ureidoJ4C (5.2, 0.05) L-Glutamic acid-U-i4C (0.04, 0.08)* L-Glutamine-UJ4C (2.4, O-04) Glycine-lJ4C (3.2, O.OS)* Glycine-2-i*C (1.3, 0.05) Hydroxyproline-2J4C (O-35, O.OS)* c+Ketoglutaric acid, sodium-5J4C (5.4, 0.05) L-Leucine-1 J4C (1, 0.05) Octanoate, sodium-l-W (1.4, 0.05) * DL-Ornithine-5-14C (2, O-05) L-Phenylalanine-1-W (O-6, O-03) Pyruvate-3-i4C (2.9, 0.05) Sarcosine, methylJ4C (3.1, 0.05)*

62 66 45 26 0.3

62

23 14

Thymine, methylJ4C (2.4, 0.04) DL-Tryptophane -3-14C (0.8, 0.03) * Injected into the head.

FLIES IN 3 HR

Radiochromatogram spots listed in order of decreasing density Prolinet y-Aminobutyric acid, prolinet Arginine, urea, one unknown Aspartic acid, proline, glutamine, glutamic acid Proline, glutamine, glutamic acid Two unknowns, proline, glutamine, glutamic acid Citrulline, urea Proline, glutamine, glutamic acid Glutamine, proline, glutamic acid? Glycine, serine Glycine, proline, serine Hydroxyproline, two unknowns Proline, glutamine, glutamic acid Leucine, one unknown Two unknowns, proline, glutamine Proline, omithine, glutamic acid Tyrosine, phenylalanine One unknown, pyruvate, proline Sarcosine, serine, proline, glutamic acid?, allantoint fi-Aminoisobutyric acid, thymine Tryptophane

t Very light spot.

Heavy labelling of purines has been noted after injection of untreated flies with a mixture of glutamine and glycine labelled in either the 1 or 2 position. This evidence is consistent with the assumption that the carbon skeleton of glycine can be incorporated without cleavage into insect purines as in purines of higher organisms. Toxicities of hydrazides For purpose of comparison it may be noted that the 24 hr LD-50 dosage of DDT applied topically to flies of the NAIDM strain was 0+1-0*2 pg/fly. When aqueous solutions of convulsive hydrazides were injected into female flies of either

158

RICHARD E. CLINE AND GEORGE W. PEARCE

the NAIDM or DDT-45 strains the following 24 hr LD-50 values were observed: TCH, thiocarbohydrazide (0.3-0.6 pg/fly), thiosemicarbazide (2-3 pg), semicarbazide (4-5 pg), and carbohydrazide (10 pg). When TCH was dissolved in ZOY/, dimethylsulphoxide, 8074 benzene and applied topically the LD-50 was 0*8-1-O pg. TCH was slightly more toxic to male than to female flies. An LD-50 of 60 pg was obtained with aqueous metrazole. The action of TCH differed from DDT in several respects. Flies injected with ex erienced active convulsions for only 15-20 min, followed by TCH (0.4 pg/fly) P near quiescence with the insects remaining on their backs. On the other hand, NAIDM flies poisoned with DDT (0.4 pg/fly) exhibited excessive neuromuscular activity for 334 hr before becoming prostrate. TCH (0.4 pg/fly) also uniquely caused excessive abdominal swelling and regurgitation, and lacked the negative temperature coefficient of action of DDT. Although the toxicity of this dosage of TCH could be prevented by 30 min prior injection of 20 pg of pyridoxal The phosphate (pH 7), such a prior injection did not reverse the toxicity of DDT. tendency of TCH solutions to become yellow on standing is presumably due to oxidation since the colour did not form when an antioxidant was added. Metabolic eflects of DDT and conedsants Soluble radiometabolites extracted from flies 3 hr after injection of ‘“C-formate consisted largely of purines (uric acid and allantoin), serine, and proline along with much lesser amounts of glutamic acid and several unknowns, two of which may be From the data of Table 3 it can be seen that uric acid degradation products. treatment of the NAIDM flies with a toxic dose of DDT or dieldrin or both strains with toxic doses of convulsive hydrazides increased the uptake of formate-14C into while reducing the content of radioproline to a large degree and purines, reducing the content of radioserine to a smaller degree, if at all. Toxic doses of metrazole had little effect on metabolism, although this compound is a wellknown convulsant. An interesting strain difference is revealed by the data of Table 3. It is to be noted that purine labelling is consistently much higher in untreated flies of the resistant strain than with untreated flies of the other strain. Moreover, the resistant flies contrasted with the susceptible flies in that treatment of the former with a large but non-toxic dose of DDT significantly reduced the labelling of purines, an effect which has been essentially duplicated in numerous experiments not recorded here. These facts seem consistent with above-mentioned data regarding proline metabolism in the two strains. With all other 14C-compounds injected into NAIDM flies treated with DDT (0.4 pg/fly) or injected with the same dosage of TCH metabolic effects were similar. Thus TCH increased the yield of 14C02 from glucose-6-14C from 50 to 72 per cent and the yield from glucose-1-14C from 37 to 92 per cent. TCH like DDT accelerated the uptake of glycine-1-14C or glycine-2- 14C into purines, and increased the labelling of urea after injection of arginine, guanidino-i4C. TCH also caused depletion of labelled trehalose.

50

10

0.4

8

Water Metrazofe

Water Semicarbazide

Water Thiocarbohydrazide

Water Thiosemicarbazide 0 100

0 100

0 60

0 80

0 100

0 80

0 40

% down

610 9500

1500 13980

2940 8490

3110 3440

1170 10380

1130 12600

2820 9920

Purine (counts/ min)

flies

1920 0

3720 200

3050 260

2590 1210

2900 40

1920 350

2520 630

Proline (counts/ min)

Susceptible (NAIDM)

1790 1170

1510 380

1910 3570

2690 2990

1280 650

1560 840

1490 1380

Serine (counts/ min)

5

0.4

10

50

15

1

10

Toxin @g/fly)

o/

0 100

0 100

0 90

0 70

0 100

0 0

0 0

do&

7670 18400

6540 19940

6620 17910

4100 3960

7540 14350

7080 7440

4310 2040

Purine (counts] min)

Resistant (DDT-45)

2130 100

3650 780

4130 100

4540 4500

2550 80

2320 2750

3420 3800

Proline (counts/ min)

flies

-

540 1230

e 2

Z 3 $

8

3

s

B

1220 590 630 730

2 8 c

3 5 $

8

?Z 1 g

2 0” *1

$J

Z $ i: g

1810 3430

680 130

950 1000

1430 1230

Serine (counts/ min)

Each fly was treated topically with 1 ~1 of DDT or dieldrin in benzene or injected with aqueous solutions of other toxins; controls received pure solvent. Three hr after treatment 1 ~1 of aqueous radioformate (100,000 counts/min) was injected per fly, and 3 hr later radiometabohtes were removed by extraction and assayed to give counts/min/fly.

15

0.5

Benzene Dieldrin

Water Carbohydrazide

0.4

Toxin &g/fly)

Benzene p,p’-DDT

Treatment

TABLE ~-EFFECT OF DDT AND OTHERCONWLSANTS ON FORMATION OF RADIOFORMATE METABOLITES

160

RICHARDE. CLINE AND GEORGEW. PEARCE

Metabolic elyects in presence of azaserine In flies injected with azaserine (5 pg/fly) along with formate-14C or glycine-1-14C the principal radiometabolites detected on chromatograms were serine, glutamine, and a product with properties expected for formylglycinamide or its riboside. The latter was also obtained, although in lower yield, when DON was substituted for azaserine. Formylglycinamide ribotide is known to accumulate in pigeon liver extracts when de novo purine synthesis is inhibited by the addition of azaserine or DON (LEVENBERG et al., 1957). The presumed radioamide metabolite gave radioglycine on chromatograms after treatment with 1 N HCl at room temperature for 24 hr. The radiometabolite did not have R, values consistent with glycinamide and had higher mobilities in solvent systems than would be expected for the ribotide, and so may be presumed to be the riboside of glycinamide. The production of this ‘riboside’ was found to be stimulated in azaserine-radioformateinjected flies by treatment with either DDT or TCH, and the more toxic the treatment the greater the production of ‘riboside’. DISCUSSION TCH followed by thiosemicarbazide and semicarbazide were the most toxic of numerous hydrazides and thiourea derivatives tested in the housefly. It is interesting that the same order of potencies has been reported for the mouse, with TCH having the highest convulsant and lethal effects of 36 hydrazides tested (JENNEY and PFEIFFER, 1958). The human toxicity of such compounds would not appear to be too high since thiosemicarbazones have received extensive study in the search for tuberculostatic drugs, leading to the marketing of amithiozone, which was later supplanted by the less toxic isonicotinic acid hydrazide (LONG, 1958). The present finding of similar metabolic effects for DDT and TCH is of particular interest in connexion with the role of aromatic amines in the mode of action of such agents. The convulsive hydrazides have been shown to interfere with the function of pyridoxal phosphate, a cofactor in enzymatic decarboxylations leading to the production of neuroactive substances such as y-aminobutyric acid and serotonin (WILLIAMS and BAIN, 1961), while hydrazides such as iproniazid have been found to elevate brain levels of serotonin and epinephrine (WOOLEY, 1962). The few reports providing evidence for the existence of catechol amines and serotonin in insects have been reviewed recently (COLHOUN, 1963), and the neurotoxin which accumulates in DDT-poisoned roaches has been characterized as an aromatic amine (HAWKINS and STERNBURG, 1964). In the search for metabolic differences between the DDT-susceptible NAIDM and the resistant DDT-45 strains some interesting preliminary data have been obtained. The observed DDT-inhibition of proline degradation and purine synthesis in the resistant strain, which is the inverse of the DDT-effect in the susceptible strain, appears relevant to the resistance phenomenon. Thus it might be supposed that in the resistant fly the toxic action of DDT is hampered by DDT-inhibition of the production of neuroactive amines or toxins, which may

SIMILAREFFECTSOF DDTANDCONWLSIVEHYDRAZIDFS ON HOUSEFLYMETABOLISM 161

be presumed to be derived from proline, degraded to purines, and necessary intermediates in the toxic action. It seems unlikely that the metabolic effects induced by DDT and TCH result simply from enhanced neuromuscular activity, because such activity was much less in flies treated with a toxic dosage of TCH than with a similarly toxic dosage of DDT. Proline depletion in the roach has been reported to be as marked after treatment with the non-stimulator-y n-valone as after treatment with DDT (RAY, 1964). REFERENCES ACOSIN M., SCARAMELLIN., DINAMARCAM. L., and ARAVENAL. (1963) Intermediary carbohydrate metabolism in Triatoma infestuns (Insecta : Hemiptera)-II. The metabolism of i4C-glucose in Triatoma infestans nymphs and the effect of DDT. Comp. Biochem. Physiol. 8, 311-320. CLINE R. E. and PEARCEG. W. (1963) Unique effects of DDT and other chlorinated hydrocarbons on the metabolism of formate and proline in the housefly. Biochemistry 2, 657-662. Biogenic amines. COLHOUNE. H. (1963) The physiological significance of acetylcholine-B. Adv. Insect Physiol. 1, 35-36. CORRIGAN J. J. and KEARNSC. W. (1958) The effect of DDT poisoning on free amino acids in the hemolymph of the American cockroach. Bull. ent. Sot. Am. 4, 95. CORRIGANJ. J. and KEARNs C. W. (1963) Amino acid metabolism in DDT-poisoned American cockroaches. J. Insect Physiol. 9, 1-12. DALE W. E., GAINERT. B., HAYES W. J., and PEARCEG. W. (1963) Poisoning by DDT: Relation between clinical signs and concentration in rat brain. Science 142, 1474-1476. HATHWAYD. E., MALLINSONA., and AKINTONWAD. A. A. (1965) Effects of dieldrin, picrotoxin and telodrin on the metabolism of ammonia in brain. Biochem. J. 94, 676-686. HAWKINSW. B. and STERNBURG J. (1964) Some chemical characteristics of a DDT-induced neuroactive substance from cockroaches and crayfish. J. econ. Ent. 57, 241-247. HAYF~ W. J. (1959) Pharmacology and toxicology of DDT. In The Insecticide Dichlorodiphenyltrichloroethane and its Significance (Ed. by MULLER P.), Vol. 2, pp. 9-247. Birkhauser, BaseI. HOSEIN E. A. (1963) The isolation of y-butyrobetaine, crotonbetaine and carnitine from brains of animals killed during induced convulsions. Arch. Biochem. Biophys. 100, 32-35. JEFFAY H. and ALVAREZJ. (1961) Liquid scintillation counting of carbon-14. Analyt. Chem. 33, 612-615. JENNFY E. H. and PFFIFWR C. C. (1958) The convulsant effect of hydrazides and the antidotal effect of anticonvulsants and metabolites. J. Pharmac. exp. Ther. 122, 110-123. KINARD F. E. (1957) Liquid scintillator for the analysis of tritium in water. Rev. scient. Instrum. 28, 293-294. LEVENBERCB., MELNICK I., and BUCHANANJ. M. (1957) Biosynthesis of the purines. J. biol. C&m. 225, 163-176. LONG E. R. (1958) The Chemistry and Chemotherapy of Tuberculosis. Williams and Wilkins, Baltimore. PERRY A. S. (1964) The physiology of insecticide resistance by insects. In Physiology of Insecta (Ed. by ROCK~TEINM.), Vol. 3, pp. 285-378. Academic Press, New York. RAY J. W. (1964) The free amino acid pool of the cockroach (Peri$Zaneta amtiana) central nervous system and the effect of insecticides. J. Insect Physiol. 10, 587-597.

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RICHARDE. CLINE AND GEORGEW. PEARCE

WILLIAMS H. L. and BAIN J. A. (1961) Convulsive effect of hydrazides: relationship to pyridoxine. In International Re&ezo of Neurobiology (Ed. by PFEIFFERC. and SMYTHIESJ.), Vol. 3, pp. 319-348. Academic Press, New York. WINTERINGHAM F. P. W. (1958) Comparative aspects of insect biochemistry with particular reference to insecticidal action. Proc. 4th int. Congr. Biochem. 12, 201-210. WINTERINGHAMF. P. W. and HARRISONA. (1956) Study of anticholinesterase action in insects by a labelled pool technique. Nature, Lond. 178, 81-83. WITTERR. F. and FARRIORW. L. (1964) Effects of dieldrin or DDT in oivo on alpha-alanine, gamma-aminobutyrate, glutamine, and glutamate in rat brain. Proc. Sot. exp. Biol. Med. 115, 487-490. WOOLEYD. W. (1962) The Biochemical Bases of Psychoses. Wiley, New York.