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Biochemical mode of action of tricyclohexylhydroxytin
Comp. gen. Pharmac., I972, 3, I25-I33. [Scientechnica (Publishers) Ltd.]
I25
BIOCHEMICAL MODE OF ACTION OF TRICYCLOHEXYLHYDROXYTIN* S A M I AHMAD]" AND C H A R L E S O. K N O W L E S Department of Entomology, University of Missouri, Columbia, Missouri 652ox , U.S.A. (Received 22 October, 197i ) ABSTRACT i. The biochemical mode of action of the acaricide tricyclohexylhydroxytin (Plictran) was studied in mice, houseflies (Musca domestica L.), and two-spotted spider mites (Tetranychus urticae Koch). 2. Plictran inhibited mouse liver and housefly thorax mitochondrial magnesiumstimulated ATPase in vitro and in vivo, and it was suggested that its mode of action in these two organisms was due to blockage of ATPase or to interference with closely related phenomena. 3. ATP dephosphorylation was observed with the 2o,ooo g particulate fraction prepared from whole homogenates of two-spotted spider mites. Hydrolysis of ATP by this preparation was stimulated by magnesium and inhibited by oligomycin and azide but not by Plictran under our assay conditions.
TRISUBSTITUTED organotin compounds are lethal to a variety of organisms including bacteria and fungi (van der Kerk and Luijten, i954; Kaars Sijpesteijn, Rijkens, Luijten, and Willemsems, t962), parasitic worms (Kerr and Walde, I956), higher plants (Hartel, I962), insects (Blum and Bower, t957; Hueck and Luijten, i958; Blum and Pratt, x96o; Gardiner and Poller, I963; Pieper and Casida, t965) , and m a m m a l s (Caujolle, Lesbre, and Meynier, i954; Stoner, Barnes, and Duff, i955; Barnes and Stoner, t958 ). Mechanism of action studies have revealed that trialkyltin compounds inhibit A T P phosphohydrolase (E.C. 3.6. I. 4, ATPase) of m a m m a l i a n liver mitochondria as well as several other processes associated with oxidative phosphorylation (Aldridge and Cremer, x955; Aldridge, 1958; Aldridge and Threlfall, t 961 ; Aldridge and Street, I964). Pieper and Casida (i965) showed that housefly, Musca domestica L., * Contribution from the Missouri Agricultural Experiment Station, Columbia. Journal series No. 6217. t Present address: Department of Entomology and Economic Zoology, Rutgers University, New Brunswick, New Jersey o89o3, U.S.A..
ATPases were inhibited by several trisubstituted organotin chlorides. Moreover, for most compounds a good correlation was noted between potency for in vitro inhibition and housefly toxicity. Tricyclohexylhydroxytin, or Plictran, is an organotin acaricide with an interesting spectrum of activity. It is effective against m a n y phytophagous mites on deciduous and citrus fruit trees, ornamentals, and greenhouse plants, and indications are that it is not harmful to predaceous mites (Gray, I968 ). Since certain trisubstituted organotin compounds are known to inhibit ATPases in m a m m a l s and houseflies, it seemed plausible that blockage of ATPase might be associated with the action of this acaricide. Therefore, we studied the effects of Plictran on ATPase preparations from mice, houseflies, and twospotted spider mites, Tetranychus urticae Koch. T h e fungicide triphenylhydroxytin or DuT e r was used for comparative purposes because of its structural resemblance to Plictran, as illustrated in Fig. x. MATERIALS AND METHODS EXPERIMENTAL ORGANISMS AND COMPOUNDS
White male mice of the Swiss-Webster strain were used; the average weight was approximately
126
AHMAD AND KNOWLES
20 g. Female houseflies of the CSMA strain were used for toxicity studies, and a random collection of male and female flies was used for enzyme inhibition studies. In each case 4-6-dayold flies were selected; individual flies weighed approximately 20 mg. Two-spotted spider mites
PLICTlxL2N (Acaricide)
FIG. I .-The
structures
of tricyclohexylhydroxytin
were reared in the laboratory on lima bean seedlings; mites were harvested by a gentle brushing of the heavily infested leaves. Individual mites weighed approximately IO ug. Plictran was supplied by the Dow Chemical Company, Midland, Michigan, and Du-Ter was obtained from Thompson-Hayward Chemical Company, Kansas City, Missouri. Oligomycin [a mixture of A (I 5 per cent) and B (75 per cent)] and analytical grade adenosine triphosphate disodium salt (ATP) were obtained from Sigma Chemical Company, St. Louis, Missouri; sodium azide was from the Fisher Chemical Company, St. Louis, Missouri. BIOASSAY Male mice were injected intraperitoneally with various concentrations of Plictran or Du-Ter, uniformly suspended in 0.2 ml. of corn oil. Four mice were treated at each dosage level, and each treatment was replicated four times. Mortality was recorded at 24 and 48 hours after treatment. Houseflies were treated topically on the prothorax with I-pl. aliquots of a methyl cellosolve : acetone mixture (2 : 3 v/v) containing various concentrations of Plictran or Du-Ter. Under these conditions Plictran formed a solution and Du-Ter a stable suspension. Twenty flies were treated at each dosage level, and each treatment was replicated four times. Mortality was recorded at 24 and 48 hours post-treatment. For mite toxicity studies the slide-dip technique (Voss, 1961; Dittrich, 1969) was used. Twenty mites were affixed to a strip of tape attached to a microscope slide. The slide was dipped and shaken for 5 seconds in an aqueous emulsion (0.2 per cent Triton X-100) of Plictran or Du-Ter. Forty mites were used at each dosage level, and each treatment was replicated twice. After treatment the slide containing the treated mites was placed on a wet sponge to maintain a high level of humidity. At 24 and 48 hours post-treatment the
Camp. gen. Pharmac.
appendages of the mites were touched with a fine needle, and those mites not responding were considered to be dead. Mortality data are expressed as intraperitoneal LD,, and topical LD,, for mice and houseflies respectively. Mite toxicity is expressed as contact
(Fungicide) (Plictran)
and triphenylhydroxytin
(Du-Terj.
LC,, or that concentration of organotin emulsion yielding 50 per cent mortality. Symptoms of organotin poisoning were recorded when apparent. ATPASE PREPARATION Freshly dissected mouse liver (I g.) was homogenized in Tris-HCl (0.005 M, &H 7.0) buffered sucrose solution (0.25 M) with a glass homogenizer fitted with a Teflon pestle. The 20 per cent (w/v) homogenate was centrifuged at 600 g for IO minutes and the particulate fraction was discarded. The 600 g supernatant was centrifuged at 20,000 g for 30 minutes, and the soluble fraction was discarded. The 20,000 g particulate was resuspended and diluted with the buffer to a concentration of 2 per cent, and 0.2 ml. was used as the mouse liver ATPase source. Thoraces from IOO houseflies, after removal of wings and legs, were homogenized in sufficient buffer to yield a 20 per cent (w/v) homogenate. The homogenate was fractionated and diluted as described for the mouse tissue. Two-spotted spider mites (450 mg.) were homogenized in buffer to yield a 4 per cent homogenate, and centrifuged as described above. Although the 20,000 g particulate fraction was used in most cases, some experiments were carried out with the 600 g particulate and 20,000 g soluble fractions. The mite fractions were not diluted; therefore, the ATPase source consisted of 0.2 ml. of the respective centrifugal fractions at the concentration of the original homogenate (4 per cent). Protein concentration in the various centrifugal fractions was determined by the Miller (1958) modification of the method of Lowry, Rosebrough, Farr, and Randall (1951). ATPASE ASSAY The incubation mixture 0.2 ml. of the appropriate
usually consisted of enzyme preparation,
ACTION OF TRICYCLOHEXYLI-IYDROXYTIN
I972, 3
0"4 ml. of Tris-glycine buffer (0"2 M, pH 8"3), 0"2 ml. of magnesium chloride (0"02 M), and o.2 ml. of ATP (o.o4 M). The final molarities of the various constituents were 8 × io -z for Trisglycine, 4 × io -3 for magnesium chloride, and 8 × lO-3 for ATP; the molar ratio of ATP to
I27
to the incubation flask, and inhibition was assessed as described above. Inhibition data are expressed as the percentage inhibition or as pIs0, which is the negative logarithm of the molar inhibitor concentration yielding 5° per cent inhibition of the enzyme.
Table L~ToxIcITY OF PLICTRANAND Du-TER TO MICE, HOUSEFLIES,AND SPIDER MITES TOXlClTY (p.p.m.) ORGANISM
ROUTE
COMPOUND 24Houm
48Hou~
Mouse
Intraperitoneal LDs0
Plictran Du-Ter
25oo 39oo
66i 7oo
Housefly
Topical LD~0
Plictran Du-Ter
64o IlOO
63o 95 °
Spider mite
Contact LCso
Plictran Du-Ter
285 iooo
15o 355
magnesium chloride was maintained at 2 : I in all experiments when this activator was used. The mixture was incubated for 3° minutes at 37° C., and the reaction was terminated with 2 ml. of sulphuric acid (7"5 per cent). A 1.5-ml. aliquot was analysed for inorganic phosphate (Pl) according to the method of Fiske and Subbarow (I925). Prior to the addition of ammonium molybdate and I-amino-2-naphthol-4-sulfonic acid the proteins were precipitated with trichloroacetic acid. The absorbance was read at 660 rim. on a Beckman DB spectrophotometer; cells of l-cm. light path were used. Controls were analysed to correct for the presence of endogenous phosphate in the enzyme preparation and for any non-enzymatic hydrolysis of ATP. Two replicates of duplicate analyses were performed for each experiment. INHIBITIONOF ATPASE ACTIVITYin vitro Appropriate concentrations of Plictran and DuTer were prepared in methylcellosolve :acetone solution (4 : i, v/v). Five gl. of the solution were added to the incubation flask and the acetone was gently evaporated leaving the methylcellosolve. (Preliminary experiments indicated that methylcellosolve at this level did not interfere with the ATPase assay.) Enzyme preparation, magnesium chloride, and buffer were added, and the mixture was shaken vigorously on a Vortex mixer. Then the mixture was preincubated for 3° minutes at 37° C. after which time the ATP was added. Following another 3o-minute incubation period at 37° C. the Pi was determined as described previously. Oligomycin and sodium azide solutions were prepared in 95 per cent ethanol and water respectively. Ten ~I. of these solutions were added
INHIBITIONOF ATPAsE ACTIVITYin vivo Mice were injected intraperitoneaUy and houseflies were treated topically with LDs0 doses of Plictran. At various post-treatment intervals 2o,ooo g particulate fractions were prepared from homogenates of mouse liver and housefly thoraces, and the ATPase activity was measured as mentioned previously. RESULTS TOXICITY STUDIES
Table I gives the toxicity of P l i c t r a n a n d D u - T e r to mice, houseflies, a n d two-spotted spider mites. Both c o m p o u n d s were of low acute toxicity to mice a n d houseflies. D u - T e r showed m o d e r a t e toxicity to spider mites, especially at 48 hours, while P l i c t r a n was quite toxic to mites with 24- a n d 4 8 - h o u r c o n t a c t LCs0 values of 285 a n d 15o pg. per g. respectively. Observations m a d e on Plictran-poisoned organisms i n d i c a t e d that it was a relatively slow-acting toxicant. Prior to d e a t h mice b e c a m e cyanotic a n d prostrate; however, n o hyperexcitability was observed. No discernible symptoms of P l i c t r a n poisoning preceeding a m o r i b u n d state were a p p a r e n t with houseflies a n d spider mites. INI-IIBITION OF MOUSE LIVER ATPAsE in vitro Fig. 2 shows the d o u b l e reciprocal plot of A T P hydrolysis b y mouse liver m i t o c h o n d r i a l
~28
Comp. gen. Pharmac.
AHMAD AND KNOWLES
magnesium-stimulated ATPase. T h e observed velocity was 3"5 pmoles o f Pi l i b e r a t e d p e r rag. p r o t e i n p e r m i n u t e . T h e m a x i m u m
velocity was 6. 7 pmoles o f Pt f o r m e d p e r mg. p r o t e i n p e r m i n u t e , a n d the K m was 6. 3 × zo - 3 M . P l i c t r a n a n d o l i g o m y e i n were
25
100
2o
0
75
~"
•
•
.._,._~ 50
IO
o~ 25
0 o
500 I / Is]
FIG. 2.--Double reciprocal plot for ATP dephosphorylation by mouse liver mitochondrial magnesium-stimulated ATPase. O, No inhibitor; A , with 7 × Io-V M Plictran; O, with i. 5 × io -8 M oligomycin.
I 10
0
1o00
I 20
I 30
activity when incubated with three different concentrations of Plictran. O, Io -v M ; n , zo-6 M ; O, Io-5 M.
75 eo
e m e l
. m
B
50pl 5 0 6.2
C
25-
0
9
1 50
Time, rain FIG. 3.--Effect of time on mouse liver A T P ~ e
1 O0 -
.a
I 40
I
I
I
,J
8
7
6
5
pl FIo. 4.--Inhibition of mouse liver ATPase by Plictran.
I29
ACTION OF TRIGYCLOHEXYLHYDROXYTIN
I972, 3
potent inhibitors of A T P hydrolysis at concentrations of 7 × Io-7 M and 1.5 × x°-S M respectively. Fig. 3 shows the effects of time on mouse liver ATPase activity when incubated with three different concentrations of Plictran.
T h e m a x i m u m velocity was 7" I gmoles of P1 formed per mg. protein per minute, and the Km was 6.5× I o - a M . Inhibition of A T P hydrolysis by oligomycin and Plictran (Fig. 6) strongly resembled that observed for the mouse liver ATPase (Fig. 2). pIso values 25-
100 20
75 Ill 0
.~
o15 50
•
m
pl 5 0 6.1 25
0
I
9
8
/ 7
--10
I
6
5
pl FIG. 5.--Inhibition of mouse liver ATPase by Du-Ter. M a x i m u m inhibition occurred at 3 ° minutes, therefore we selected this interval as the incubation time for additional inhibition studies. Also, it can be noted that inhibition increased sharply when the Plictran concentration was increased from 10 .7 M to t o -e M. However, when the Plictran concentration was increased further to Io -5 M a marked decrease in inhibition was apparent. T h e PIs0 values for inhibition of A T P hydrolysis by the mouse liver preparation were 6-2 for Plictran (Fig. 4) and 6.i for Du-Ter (Fig. 5). INHIBITION OF HOUSEFLY THORAX ATPASE
in
vitro T h e Lineweaver-Burk plot of A T P de~ phosphorylation by housefly thoracic mitochondrial magnesium-stimulated ATPase is presented in Fig. 6. This enzyme liberated 4"2 gmoles of Pi per mg. protein per minute.
0
500 1000 I / I~l FIG. 6.--Double reciprocal plot for ATP dephosphorylation by housefly thorax mitochondrial magnesium-stimulated ATPase. O, No inhibition; A, with 7 × Io-7 M Plictran; O, with i- 5 × io -8 M oligomycin. were also similar to those found with the mouse liver enzyme, being 6.2 for Plictran (Fig. 7) and 6.0 for Du-Ter (Fig. 8). INHIBITION OF SPIDER MITE ATPAsE
in vitro
An approximate parallelism was observed between mouse and housefly ATPases, both in terms of A T P hydrolysis and reaction with oligomycin, Plictran, and Du-Ter. However, a different situation was found in the case of two-spotted spider mites. A double reciprocal plot for the hydrolysis of A T P by the 2o,0oo g particulate fraction prepared from a whole-mite homogenate revealed a maxim u m velocity of o.6 gmole of inorganic phosphate formed per mg. protein per minute, and a K,, of 2"3 × IO-3 M (Fig. 9). The actual observed rate of formation of Pl was o.42 gmole per mg. protein per minute.
Comp. gen. Pharmac.
AHMAD AND KNOWLES
z3o
Although oligomycin inhibited A T P hydrolysis to some extent, a more significant observation was that no inhibition of A T P hydrolysis was apparent in the presence of either Plictran or Du-Ter. Therefore, we performed additional experiments to understand better the nature of two-spotted spider mite
ATPases, and the results are given in Table II. Hydrolysis of A T P was obtained with the 6o:)g particulate, 2o,ooog particulate, and 2o, o o o g soluble fractions with the rate decreasing in the same order. Magnesium ions activated A T P hydrolysis by the 2o0oo g particulate and soluble fractions but
1 O0
100
75
75
eo
c 0
om .ll .m
* J
.0
50
,m
e-
pl 50 6.2 25
50 p150 6.0
m
25
0
8
t
I
1
I
7
6
5
4
0
9
pl
I
i
I
,I
8
7
6
5
pl
FIO. 7.--Inhibition of housefly thorax ATPase by Plictran.
FIG. &--Inhibition of housefly thorax ATPase by Du-Ter.
10
I/v 5
0
I
I
500
1000
1i FIo. 9.--Double reciprocal plot for ATP dephosphorylation by two-spotted spider mite 20,000 g particulate magnesium-stimulated ATPase.
I972, 3
ACTION OF TRICYCLOHEXYLHYDROXYTIN
inhibited A T P hydrolysis by the 6oog particulate fraction. Azide inhibited hydrolysis by all three fractions. Oligomycin inhibited ATP hydrolysis by the 2o,ooog particulate and soluble fractions but apparently activated the hydrolysis of ATP by the 6oog particulate fraction. There was no
I3I
Pieper and Casida (1965), working with housefly thorax particulate (23,500 g) ATPase from a SCR strain, reported a value of 4"7 gmoles per mg. protein per minute for A T P dephosphorylation and a /(,, of 3-8 X Io -a M. They also reported a pIs0 value of 7"0 for the inhibition of A T P hydrolysis by
Table//.--NATURE OF TWO-SPOTTED SPIDER MITE ATPAsES
SUBCELLULAR FRACTION
Particulate, 6oo g Particulate, 2o,ooo g Supernatant, 2o0oo g
El
(nmoles per rag. per minute)
PERCENTAGE ACTIVATION ( + ) OR INHIBITION (--)
Mg 2+ (4 × I°-3 M)
(i z Io-' M)
68I 42o
--29 +5 °
--6 4 --6 9
+24
173
+II 7
--79
-23
detectable inhibition of ATP hydrolysis by Plictran or Du-Ter in the concentration range of Io-S-Io -5 M by any of the three fractions. INHIBITION OF MOUSE LIVER AND HOUSEFLY
THORAX ATPAsE in vivo Since Plictran inhibited mouse and housefly ATPase in vitro, an attempt was made to ascertain whether ATPase was inhibited in vivo in animals treated with this acaricide. No ATPase inhibition was detected in mice and houseflies throughout 8 hours posttreatment. By 24 hours inhibition was 31 per cent in mice and 13 per cent in houseflies. ATPase assay of surviving animals at 36 hours after treatment revealed 15 per cent and 12 per cent inhibition for mice and houseflies respectively. DISCUSSION Plictran is a very insoluble compound; in fact it is virtually insoluble in water ( < o . o o o i g. per Ioo ml.) and has a very low order of solubility in most organic solvents (Gray, 1968). Thus, the decline in inhibition of A T P dephosphorylation observed in the present study (Figs. 3, 4, 7) at high concentrations of Plictran ( > IO - 6 M ) is attributed largely to its solubility properties.
NaNa
Oligomycin (3 × IO-S M --31
triphenyltin chloride. The 48-hour LDs0 for topically applied triphenyltin chloride was 54 Pg. per g. (Pieper and Casida, I965). With two exceptions our data compare favourably with those of Pieper and Casida (I965). The rate of dephosphorylation for our housefly (CSMA strain) thorax particulate (2o000 g) ATPase was 4"2 pmoles of P~ per mg. protein per minute, and the K m was 6. 3 × Io -3 M. However, we calculated a pls0 value of 6.0 for Du-Ter (triphenylhydroxytin), and the 48-hour LDs0 of topically applied Du-Ter was 95 ° ~tg. per g. Thus, Du-Ter is IO times less potent in vitro and 17.6 times less toxic than triphenyltin chloride. This apparent disparity is probably due to the different strains of houseflies used in the two studies and to the different anionic groups linked to the organotin radical, the latter possibly being the more significant. Aldridge and Threlfall (I 96 I) showed that triethyltin inhibited dinitrophenol- and magnesium-stimulated ATPase, as well as the ATPJ~p~ exchange and phosphorylating respiration. In view of these facts Pieper and Casida (i 965) suggested that ATPase inhibition could be used as an index for the interference of trialkyltins with several related processes. Thus, from the present study it
~32
AHMAD AND KNOWLES
seems possible that the action of Plictran, a cycloalkyltin, a n d of D u - T e r , a triaryltin, could be due to blockage of the A T P a s e or to interference with these closely related p h e n o m e n a in the mouse a n d housefly. However, there was no evidence to suggest that the m o d e of acaricidal activity of Plictran was related to inhibition of A T P hydrolysis by two-spotted spider mite ATPases assayed under our conditions. T h e e n z y m e preparation from spider mites consisted of centrifugal fractions of whole homogenates; therefore, it seems logical to predict that m o r e than one A T P hydrolysing e n z y m e was p r o b a b l y present. Moreover, based on oligomycin and azide sensitivity and m a g n e s i u m stimulation, it is likely that at least some of the A T P hydrolysis of spider mite mitochondrial fraction (2o,ooo g particulate) was catalysed by A T P phosphohydrolase (E.C. 3.6. x. 4). Thus, the failure of Plictran and D u - T e r to inhibit A T P dephosphorylation, at least partially, was an unexpected result, especially since inhibition was obtained with the mouse liver and housefly thorax mitochondrial ATPases. T h e r e are several possible reasons w h y inhibition of A T P dephosphorylation by Plictran and D u - T e r was not observed. According to Aldridge and Street (i964) a n d Aldridge and Threlfall (i961) rat liver mitochondria are capable of extracting tributyltin from the m e d i u m , and triethyhin has an affinity for lipophilic and negatively charged substances. Therefore, the Plictran a n d D u - T e r m a y have been ' b o u n d ' with lipophilic components in the whole-mite h o m o g e n a t e a n d hence were unavailable to inhibit A T P dephosphorylation. Moreover, the mite A T P a s e h a d a m u c h lower specific activity based on total protein than did the mouse or housefly enzymes, a n d Pieper a n d Casida (I965) observed that the sensitivity of A T P hydrolysis to triphenyltin chloride was directly related to specific activity. A final possibility which should be considered is that two-spotted spider mites possess no Plictranor Du-Ter-sensitive ATPase. In other words the organotin ' b i n d i n g s i t e ( s ) ' is absent from spider mite ATPase(s). Clearly m o r e work is required to elucidate the precise
Comp. gen. Pharmac.
m e c h a n i s m of action for Plictran on twospotted spider mites. REFERENCES ALDRIDO~, W. N. (i958), ' T h e biochemistry of organotin compounds. Trialkyltins and oxidative phosphorylation ', Biochem. ft., 69, 367376. ALDRIDOE, W. N., and CREM~R, J. E. (i955) , 'Biochemistry of organotin compounds. Diethyltin dichloride and triethyhin sulfate ', Biochem. 07., 6I, 4o6-418. ALDRIDCE, W. N., and STREET, B. S. (I964), ' Oxidative phosphorylation. Biochemical effects and properties of trialkytins ', Biochem. J., 9x, 287-297. ALDRIDC~, W. N., and TnRELFALL, C. J. (i96i), 'Trialkyltins and oxidative phosphorylation. The (3~p) phosphate adenosine triphosphate exchange reaction ', Biochem. 07., 79, 2 [4-219. BARNES,J. M., and STONER, H. B. (i958), ' Toxic properties of some dialkyl and trialkyl tin salts ', Br. aT. ind. Med., 15, I5-22. BLUM, M. S., and BOWF.R, F. A. ([957), ' T h e evaluation of triethyltin hydroxide and its esters as insecticides ', J. econ. EAt., 5o, 84-86. BLUM, M. S., and PRATT, J. J., jun. ([960), 'Relationship between structure and insecticidal activity of some organotin compounds ', ft. econ. EAt., 53, 445-448. CAVJOLLE, F., LESBR~, M., and MEYNIER, D. (I954) , ' Sur la toxicitd du tetramethylstannane et du tetraethylstannane ', C. r. hebd. Sdanc. Acad. Sci., Paris, 239, 556-558. DITTRmn, V. (I969), ' Chlorphenamidine negatively correlated with OP resistance in a strain of two-spotted spider mite ', .7. econ. EAt., 62, 44-47. FIs~E, C. H., and SUBBAROW, Y. (I925) , ' T h e colorimetric determination of phosphorus ', .7. biol. Chem., 66, 375-4oo. GARDINER, B. G., and POLLER, R. C. ([963), 'Insecticidal activity and structure of some organotin compounds ', Bull. ent. Res., 55, [ 7-2 I. GRAY, H. E. (I968), 'Plietran miticide--new approach to mite control ', Down to Earth, "3, 3-5HART[L, K. (I962), ' Triphenyl tin compounds ', Agric. vet. Chem., 3, [9-24. HUECK, H. J., and LUIJTEN, J. G. A. ([958), 'Organo-tin compounds as textile preservatives ', 07. Soc. Dyers Colourists, 74, 476-48o. KAARS SIJPESTEIJN, A., RIJKENS, F., LUIJTEN, J. G. A., and WILL~MSENS, L. C. ([962), ' On the antifungal and antibacterial activity of some trisubstituted organogermanium, organotin and organolead compounds ', Antonie van Leeuwenhoek, 28, 346-356. KERR, K. B., and WALDE, A. W. (I956), ' T h e anthelmintic activity of tetravalent tin compounds ', Expl. Parasit., 5, 56o-57 °.
1972, 3
ACTION OF TRICYCLOHEXYLHYDROXYTIN
LowRy, O. H., ROSEBROUGn,N. J., FARR, A. L., and RANDALL,R. J. (x95Q, ' Protein measurement with the Folin phenol reagent ', 07. biol. Chem., x93, 265-275. MmLER, G. L. (x959), ' Protein determination for large numbers of samples ', Analyt. Chem., 3 x, 964 • ProPER, G. R., and CASXDA,J. E. (1965), ' Housefly adenosine triphosphatases and their inhibition by insecticidal organotin compounds ', 07. econ. Ent., 58, 392-4oo. STONER, H. B., BARNES,J. M., and DuFf, J. I. (I955), 'Studies on the toxicity of alkyl tin compounds ', Br. 07. Pharmac., xo, I6-25.
I33
VAN DER KERK, G. J. M., and LUIJTEN, J. G. A. (1954) , 'Investigations on organo-tin compounds. III. The biocida|properties oforganotin compounds ', aT. appl. Chem., 'b 315-319Voss, G. ( 196 I), ' Ein neues Akarizid-Austestungsverfahren ftir Spinnmilben ', Anz. Schiidlingsk, 34, 76-77 •
Key Word Index: ATPase, Du-Ter, Plictran, tricyclohexylhydroxytin, triphenylhydroxytin, mouse, housefly, Musca domestica L., two-spotted spider mite, Tetranychus urticae Koch, spider mite ATPase, acaricides, organotin compounds.
I25
BIOCHEMICAL MODE OF ACTION OF TRICYCLOHEXYLHYDROXYTIN* S A M I AHMAD]" AND C H A R L E S O. K N O W L E S Department of Entomology, University of Missouri, Columbia, Missouri 652ox , U.S.A. (Received 22 October, 197i ) ABSTRACT i. The biochemical mode of action of the acaricide tricyclohexylhydroxytin (Plictran) was studied in mice, houseflies (Musca domestica L.), and two-spotted spider mites (Tetranychus urticae Koch). 2. Plictran inhibited mouse liver and housefly thorax mitochondrial magnesiumstimulated ATPase in vitro and in vivo, and it was suggested that its mode of action in these two organisms was due to blockage of ATPase or to interference with closely related phenomena. 3. ATP dephosphorylation was observed with the 2o,ooo g particulate fraction prepared from whole homogenates of two-spotted spider mites. Hydrolysis of ATP by this preparation was stimulated by magnesium and inhibited by oligomycin and azide but not by Plictran under our assay conditions.
TRISUBSTITUTED organotin compounds are lethal to a variety of organisms including bacteria and fungi (van der Kerk and Luijten, i954; Kaars Sijpesteijn, Rijkens, Luijten, and Willemsems, t962), parasitic worms (Kerr and Walde, I956), higher plants (Hartel, I962), insects (Blum and Bower, t957; Hueck and Luijten, i958; Blum and Pratt, x96o; Gardiner and Poller, I963; Pieper and Casida, t965) , and m a m m a l s (Caujolle, Lesbre, and Meynier, i954; Stoner, Barnes, and Duff, i955; Barnes and Stoner, t958 ). Mechanism of action studies have revealed that trialkyltin compounds inhibit A T P phosphohydrolase (E.C. 3.6. I. 4, ATPase) of m a m m a l i a n liver mitochondria as well as several other processes associated with oxidative phosphorylation (Aldridge and Cremer, x955; Aldridge, 1958; Aldridge and Threlfall, t 961 ; Aldridge and Street, I964). Pieper and Casida (i965) showed that housefly, Musca domestica L., * Contribution from the Missouri Agricultural Experiment Station, Columbia. Journal series No. 6217. t Present address: Department of Entomology and Economic Zoology, Rutgers University, New Brunswick, New Jersey o89o3, U.S.A..
ATPases were inhibited by several trisubstituted organotin chlorides. Moreover, for most compounds a good correlation was noted between potency for in vitro inhibition and housefly toxicity. Tricyclohexylhydroxytin, or Plictran, is an organotin acaricide with an interesting spectrum of activity. It is effective against m a n y phytophagous mites on deciduous and citrus fruit trees, ornamentals, and greenhouse plants, and indications are that it is not harmful to predaceous mites (Gray, I968 ). Since certain trisubstituted organotin compounds are known to inhibit ATPases in m a m m a l s and houseflies, it seemed plausible that blockage of ATPase might be associated with the action of this acaricide. Therefore, we studied the effects of Plictran on ATPase preparations from mice, houseflies, and twospotted spider mites, Tetranychus urticae Koch. T h e fungicide triphenylhydroxytin or DuT e r was used for comparative purposes because of its structural resemblance to Plictran, as illustrated in Fig. x. MATERIALS AND METHODS EXPERIMENTAL ORGANISMS AND COMPOUNDS
White male mice of the Swiss-Webster strain were used; the average weight was approximately
126
AHMAD AND KNOWLES
20 g. Female houseflies of the CSMA strain were used for toxicity studies, and a random collection of male and female flies was used for enzyme inhibition studies. In each case 4-6-dayold flies were selected; individual flies weighed approximately 20 mg. Two-spotted spider mites
PLICTlxL2N (Acaricide)
FIG. I .-The
structures
of tricyclohexylhydroxytin
were reared in the laboratory on lima bean seedlings; mites were harvested by a gentle brushing of the heavily infested leaves. Individual mites weighed approximately IO ug. Plictran was supplied by the Dow Chemical Company, Midland, Michigan, and Du-Ter was obtained from Thompson-Hayward Chemical Company, Kansas City, Missouri. Oligomycin [a mixture of A (I 5 per cent) and B (75 per cent)] and analytical grade adenosine triphosphate disodium salt (ATP) were obtained from Sigma Chemical Company, St. Louis, Missouri; sodium azide was from the Fisher Chemical Company, St. Louis, Missouri. BIOASSAY Male mice were injected intraperitoneally with various concentrations of Plictran or Du-Ter, uniformly suspended in 0.2 ml. of corn oil. Four mice were treated at each dosage level, and each treatment was replicated four times. Mortality was recorded at 24 and 48 hours after treatment. Houseflies were treated topically on the prothorax with I-pl. aliquots of a methyl cellosolve : acetone mixture (2 : 3 v/v) containing various concentrations of Plictran or Du-Ter. Under these conditions Plictran formed a solution and Du-Ter a stable suspension. Twenty flies were treated at each dosage level, and each treatment was replicated four times. Mortality was recorded at 24 and 48 hours post-treatment. For mite toxicity studies the slide-dip technique (Voss, 1961; Dittrich, 1969) was used. Twenty mites were affixed to a strip of tape attached to a microscope slide. The slide was dipped and shaken for 5 seconds in an aqueous emulsion (0.2 per cent Triton X-100) of Plictran or Du-Ter. Forty mites were used at each dosage level, and each treatment was replicated twice. After treatment the slide containing the treated mites was placed on a wet sponge to maintain a high level of humidity. At 24 and 48 hours post-treatment the
Camp. gen. Pharmac.
appendages of the mites were touched with a fine needle, and those mites not responding were considered to be dead. Mortality data are expressed as intraperitoneal LD,, and topical LD,, for mice and houseflies respectively. Mite toxicity is expressed as contact
(Fungicide) (Plictran)
and triphenylhydroxytin
(Du-Terj.
LC,, or that concentration of organotin emulsion yielding 50 per cent mortality. Symptoms of organotin poisoning were recorded when apparent. ATPASE PREPARATION Freshly dissected mouse liver (I g.) was homogenized in Tris-HCl (0.005 M, &H 7.0) buffered sucrose solution (0.25 M) with a glass homogenizer fitted with a Teflon pestle. The 20 per cent (w/v) homogenate was centrifuged at 600 g for IO minutes and the particulate fraction was discarded. The 600 g supernatant was centrifuged at 20,000 g for 30 minutes, and the soluble fraction was discarded. The 20,000 g particulate was resuspended and diluted with the buffer to a concentration of 2 per cent, and 0.2 ml. was used as the mouse liver ATPase source. Thoraces from IOO houseflies, after removal of wings and legs, were homogenized in sufficient buffer to yield a 20 per cent (w/v) homogenate. The homogenate was fractionated and diluted as described for the mouse tissue. Two-spotted spider mites (450 mg.) were homogenized in buffer to yield a 4 per cent homogenate, and centrifuged as described above. Although the 20,000 g particulate fraction was used in most cases, some experiments were carried out with the 600 g particulate and 20,000 g soluble fractions. The mite fractions were not diluted; therefore, the ATPase source consisted of 0.2 ml. of the respective centrifugal fractions at the concentration of the original homogenate (4 per cent). Protein concentration in the various centrifugal fractions was determined by the Miller (1958) modification of the method of Lowry, Rosebrough, Farr, and Randall (1951). ATPASE ASSAY The incubation mixture 0.2 ml. of the appropriate
usually consisted of enzyme preparation,
ACTION OF TRICYCLOHEXYLI-IYDROXYTIN
I972, 3
0"4 ml. of Tris-glycine buffer (0"2 M, pH 8"3), 0"2 ml. of magnesium chloride (0"02 M), and o.2 ml. of ATP (o.o4 M). The final molarities of the various constituents were 8 × io -z for Trisglycine, 4 × io -3 for magnesium chloride, and 8 × lO-3 for ATP; the molar ratio of ATP to
I27
to the incubation flask, and inhibition was assessed as described above. Inhibition data are expressed as the percentage inhibition or as pIs0, which is the negative logarithm of the molar inhibitor concentration yielding 5° per cent inhibition of the enzyme.
Table L~ToxIcITY OF PLICTRANAND Du-TER TO MICE, HOUSEFLIES,AND SPIDER MITES TOXlClTY (p.p.m.) ORGANISM
ROUTE
COMPOUND 24Houm
48Hou~
Mouse
Intraperitoneal LDs0
Plictran Du-Ter
25oo 39oo
66i 7oo
Housefly
Topical LD~0
Plictran Du-Ter
64o IlOO
63o 95 °
Spider mite
Contact LCso
Plictran Du-Ter
285 iooo
15o 355
magnesium chloride was maintained at 2 : I in all experiments when this activator was used. The mixture was incubated for 3° minutes at 37° C., and the reaction was terminated with 2 ml. of sulphuric acid (7"5 per cent). A 1.5-ml. aliquot was analysed for inorganic phosphate (Pl) according to the method of Fiske and Subbarow (I925). Prior to the addition of ammonium molybdate and I-amino-2-naphthol-4-sulfonic acid the proteins were precipitated with trichloroacetic acid. The absorbance was read at 660 rim. on a Beckman DB spectrophotometer; cells of l-cm. light path were used. Controls were analysed to correct for the presence of endogenous phosphate in the enzyme preparation and for any non-enzymatic hydrolysis of ATP. Two replicates of duplicate analyses were performed for each experiment. INHIBITIONOF ATPASE ACTIVITYin vitro Appropriate concentrations of Plictran and DuTer were prepared in methylcellosolve :acetone solution (4 : i, v/v). Five gl. of the solution were added to the incubation flask and the acetone was gently evaporated leaving the methylcellosolve. (Preliminary experiments indicated that methylcellosolve at this level did not interfere with the ATPase assay.) Enzyme preparation, magnesium chloride, and buffer were added, and the mixture was shaken vigorously on a Vortex mixer. Then the mixture was preincubated for 3° minutes at 37° C. after which time the ATP was added. Following another 3o-minute incubation period at 37° C. the Pi was determined as described previously. Oligomycin and sodium azide solutions were prepared in 95 per cent ethanol and water respectively. Ten ~I. of these solutions were added
INHIBITIONOF ATPAsE ACTIVITYin vivo Mice were injected intraperitoneaUy and houseflies were treated topically with LDs0 doses of Plictran. At various post-treatment intervals 2o,ooo g particulate fractions were prepared from homogenates of mouse liver and housefly thoraces, and the ATPase activity was measured as mentioned previously. RESULTS TOXICITY STUDIES
Table I gives the toxicity of P l i c t r a n a n d D u - T e r to mice, houseflies, a n d two-spotted spider mites. Both c o m p o u n d s were of low acute toxicity to mice a n d houseflies. D u - T e r showed m o d e r a t e toxicity to spider mites, especially at 48 hours, while P l i c t r a n was quite toxic to mites with 24- a n d 4 8 - h o u r c o n t a c t LCs0 values of 285 a n d 15o pg. per g. respectively. Observations m a d e on Plictran-poisoned organisms i n d i c a t e d that it was a relatively slow-acting toxicant. Prior to d e a t h mice b e c a m e cyanotic a n d prostrate; however, n o hyperexcitability was observed. No discernible symptoms of P l i c t r a n poisoning preceeding a m o r i b u n d state were a p p a r e n t with houseflies a n d spider mites. INI-IIBITION OF MOUSE LIVER ATPAsE in vitro Fig. 2 shows the d o u b l e reciprocal plot of A T P hydrolysis b y mouse liver m i t o c h o n d r i a l
~28
Comp. gen. Pharmac.
AHMAD AND KNOWLES
magnesium-stimulated ATPase. T h e observed velocity was 3"5 pmoles o f Pi l i b e r a t e d p e r rag. p r o t e i n p e r m i n u t e . T h e m a x i m u m
velocity was 6. 7 pmoles o f Pt f o r m e d p e r mg. p r o t e i n p e r m i n u t e , a n d the K m was 6. 3 × zo - 3 M . P l i c t r a n a n d o l i g o m y e i n were
25
100
2o
0
75
~"
•
•
.._,._~ 50
IO
o~ 25
0 o
500 I / Is]
FIG. 2.--Double reciprocal plot for ATP dephosphorylation by mouse liver mitochondrial magnesium-stimulated ATPase. O, No inhibitor; A , with 7 × Io-V M Plictran; O, with i. 5 × io -8 M oligomycin.
I 10
0
1o00
I 20
I 30
activity when incubated with three different concentrations of Plictran. O, Io -v M ; n , zo-6 M ; O, Io-5 M.
75 eo
e m e l
. m
B
50pl 5 0 6.2
C
25-
0
9
1 50
Time, rain FIG. 3.--Effect of time on mouse liver A T P ~ e
1 O0 -
.a
I 40
I
I
I
,J
8
7
6
5
pl FIo. 4.--Inhibition of mouse liver ATPase by Plictran.
I29
ACTION OF TRIGYCLOHEXYLHYDROXYTIN
I972, 3
potent inhibitors of A T P hydrolysis at concentrations of 7 × Io-7 M and 1.5 × x°-S M respectively. Fig. 3 shows the effects of time on mouse liver ATPase activity when incubated with three different concentrations of Plictran.
T h e m a x i m u m velocity was 7" I gmoles of P1 formed per mg. protein per minute, and the Km was 6.5× I o - a M . Inhibition of A T P hydrolysis by oligomycin and Plictran (Fig. 6) strongly resembled that observed for the mouse liver ATPase (Fig. 2). pIso values 25-
100 20
75 Ill 0
.~
o15 50
•
m
pl 5 0 6.1 25
0
I
9
8
/ 7
--10
I
6
5
pl FIG. 5.--Inhibition of mouse liver ATPase by Du-Ter. M a x i m u m inhibition occurred at 3 ° minutes, therefore we selected this interval as the incubation time for additional inhibition studies. Also, it can be noted that inhibition increased sharply when the Plictran concentration was increased from 10 .7 M to t o -e M. However, when the Plictran concentration was increased further to Io -5 M a marked decrease in inhibition was apparent. T h e PIs0 values for inhibition of A T P hydrolysis by the mouse liver preparation were 6-2 for Plictran (Fig. 4) and 6.i for Du-Ter (Fig. 5). INHIBITION OF HOUSEFLY THORAX ATPASE
in
vitro T h e Lineweaver-Burk plot of A T P de~ phosphorylation by housefly thoracic mitochondrial magnesium-stimulated ATPase is presented in Fig. 6. This enzyme liberated 4"2 gmoles of Pi per mg. protein per minute.
0
500 1000 I / I~l FIG. 6.--Double reciprocal plot for ATP dephosphorylation by housefly thorax mitochondrial magnesium-stimulated ATPase. O, No inhibition; A, with 7 × Io-7 M Plictran; O, with i- 5 × io -8 M oligomycin. were also similar to those found with the mouse liver enzyme, being 6.2 for Plictran (Fig. 7) and 6.0 for Du-Ter (Fig. 8). INHIBITION OF SPIDER MITE ATPAsE
in vitro
An approximate parallelism was observed between mouse and housefly ATPases, both in terms of A T P hydrolysis and reaction with oligomycin, Plictran, and Du-Ter. However, a different situation was found in the case of two-spotted spider mites. A double reciprocal plot for the hydrolysis of A T P by the 2o,0oo g particulate fraction prepared from a whole-mite homogenate revealed a maxim u m velocity of o.6 gmole of inorganic phosphate formed per mg. protein per minute, and a K,, of 2"3 × IO-3 M (Fig. 9). The actual observed rate of formation of Pl was o.42 gmole per mg. protein per minute.
Comp. gen. Pharmac.
AHMAD AND KNOWLES
z3o
Although oligomycin inhibited A T P hydrolysis to some extent, a more significant observation was that no inhibition of A T P hydrolysis was apparent in the presence of either Plictran or Du-Ter. Therefore, we performed additional experiments to understand better the nature of two-spotted spider mite
ATPases, and the results are given in Table II. Hydrolysis of A T P was obtained with the 6o:)g particulate, 2o,ooog particulate, and 2o, o o o g soluble fractions with the rate decreasing in the same order. Magnesium ions activated A T P hydrolysis by the 2o0oo g particulate and soluble fractions but
1 O0
100
75
75
eo
c 0
om .ll .m
* J
.0
50
,m
e-
pl 50 6.2 25
50 p150 6.0
m
25
0
8
t
I
1
I
7
6
5
4
0
9
pl
I
i
I
,I
8
7
6
5
pl
FIO. 7.--Inhibition of housefly thorax ATPase by Plictran.
FIG. &--Inhibition of housefly thorax ATPase by Du-Ter.
10
I/v 5
0
I
I
500
1000
1i FIo. 9.--Double reciprocal plot for ATP dephosphorylation by two-spotted spider mite 20,000 g particulate magnesium-stimulated ATPase.
I972, 3
ACTION OF TRICYCLOHEXYLHYDROXYTIN
inhibited A T P hydrolysis by the 6oog particulate fraction. Azide inhibited hydrolysis by all three fractions. Oligomycin inhibited ATP hydrolysis by the 2o,ooog particulate and soluble fractions but apparently activated the hydrolysis of ATP by the 6oog particulate fraction. There was no
I3I
Pieper and Casida (1965), working with housefly thorax particulate (23,500 g) ATPase from a SCR strain, reported a value of 4"7 gmoles per mg. protein per minute for A T P dephosphorylation and a /(,, of 3-8 X Io -a M. They also reported a pIs0 value of 7"0 for the inhibition of A T P hydrolysis by
Table//.--NATURE OF TWO-SPOTTED SPIDER MITE ATPAsES
SUBCELLULAR FRACTION
Particulate, 6oo g Particulate, 2o,ooo g Supernatant, 2o0oo g
El
(nmoles per rag. per minute)
PERCENTAGE ACTIVATION ( + ) OR INHIBITION (--)
Mg 2+ (4 × I°-3 M)
(i z Io-' M)
68I 42o
--29 +5 °
--6 4 --6 9
+24
173
+II 7
--79
-23
detectable inhibition of ATP hydrolysis by Plictran or Du-Ter in the concentration range of Io-S-Io -5 M by any of the three fractions. INHIBITION OF MOUSE LIVER AND HOUSEFLY
THORAX ATPAsE in vivo Since Plictran inhibited mouse and housefly ATPase in vitro, an attempt was made to ascertain whether ATPase was inhibited in vivo in animals treated with this acaricide. No ATPase inhibition was detected in mice and houseflies throughout 8 hours posttreatment. By 24 hours inhibition was 31 per cent in mice and 13 per cent in houseflies. ATPase assay of surviving animals at 36 hours after treatment revealed 15 per cent and 12 per cent inhibition for mice and houseflies respectively. DISCUSSION Plictran is a very insoluble compound; in fact it is virtually insoluble in water ( < o . o o o i g. per Ioo ml.) and has a very low order of solubility in most organic solvents (Gray, 1968). Thus, the decline in inhibition of A T P dephosphorylation observed in the present study (Figs. 3, 4, 7) at high concentrations of Plictran ( > IO - 6 M ) is attributed largely to its solubility properties.
NaNa
Oligomycin (3 × IO-S M --31
triphenyltin chloride. The 48-hour LDs0 for topically applied triphenyltin chloride was 54 Pg. per g. (Pieper and Casida, I965). With two exceptions our data compare favourably with those of Pieper and Casida (I965). The rate of dephosphorylation for our housefly (CSMA strain) thorax particulate (2o000 g) ATPase was 4"2 pmoles of P~ per mg. protein per minute, and the K m was 6. 3 × Io -3 M. However, we calculated a pls0 value of 6.0 for Du-Ter (triphenylhydroxytin), and the 48-hour LDs0 of topically applied Du-Ter was 95 ° ~tg. per g. Thus, Du-Ter is IO times less potent in vitro and 17.6 times less toxic than triphenyltin chloride. This apparent disparity is probably due to the different strains of houseflies used in the two studies and to the different anionic groups linked to the organotin radical, the latter possibly being the more significant. Aldridge and Threlfall (I 96 I) showed that triethyltin inhibited dinitrophenol- and magnesium-stimulated ATPase, as well as the ATPJ~p~ exchange and phosphorylating respiration. In view of these facts Pieper and Casida (i 965) suggested that ATPase inhibition could be used as an index for the interference of trialkyltins with several related processes. Thus, from the present study it
~32
AHMAD AND KNOWLES
seems possible that the action of Plictran, a cycloalkyltin, a n d of D u - T e r , a triaryltin, could be due to blockage of the A T P a s e or to interference with these closely related p h e n o m e n a in the mouse a n d housefly. However, there was no evidence to suggest that the m o d e of acaricidal activity of Plictran was related to inhibition of A T P hydrolysis by two-spotted spider mite ATPases assayed under our conditions. T h e e n z y m e preparation from spider mites consisted of centrifugal fractions of whole homogenates; therefore, it seems logical to predict that m o r e than one A T P hydrolysing e n z y m e was p r o b a b l y present. Moreover, based on oligomycin and azide sensitivity and m a g n e s i u m stimulation, it is likely that at least some of the A T P hydrolysis of spider mite mitochondrial fraction (2o,ooo g particulate) was catalysed by A T P phosphohydrolase (E.C. 3.6. x. 4). Thus, the failure of Plictran and D u - T e r to inhibit A T P dephosphorylation, at least partially, was an unexpected result, especially since inhibition was obtained with the mouse liver and housefly thorax mitochondrial ATPases. T h e r e are several possible reasons w h y inhibition of A T P dephosphorylation by Plictran and D u - T e r was not observed. According to Aldridge and Street (i964) a n d Aldridge and Threlfall (i961) rat liver mitochondria are capable of extracting tributyltin from the m e d i u m , and triethyhin has an affinity for lipophilic and negatively charged substances. Therefore, the Plictran a n d D u - T e r m a y have been ' b o u n d ' with lipophilic components in the whole-mite h o m o g e n a t e a n d hence were unavailable to inhibit A T P dephosphorylation. Moreover, the mite A T P a s e h a d a m u c h lower specific activity based on total protein than did the mouse or housefly enzymes, a n d Pieper a n d Casida (I965) observed that the sensitivity of A T P hydrolysis to triphenyltin chloride was directly related to specific activity. A final possibility which should be considered is that two-spotted spider mites possess no Plictranor Du-Ter-sensitive ATPase. In other words the organotin ' b i n d i n g s i t e ( s ) ' is absent from spider mite ATPase(s). Clearly m o r e work is required to elucidate the precise
Comp. gen. Pharmac.
m e c h a n i s m of action for Plictran on twospotted spider mites. REFERENCES ALDRIDO~, W. N. (i958), ' T h e biochemistry of organotin compounds. Trialkyltins and oxidative phosphorylation ', Biochem. ft., 69, 367376. ALDRIDOE, W. N., and CREM~R, J. E. (i955) , 'Biochemistry of organotin compounds. Diethyltin dichloride and triethyhin sulfate ', Biochem. 07., 6I, 4o6-418. ALDRIDCE, W. N., and STREET, B. S. (I964), ' Oxidative phosphorylation. Biochemical effects and properties of trialkytins ', Biochem. J., 9x, 287-297. ALDRIDC~, W. N., and TnRELFALL, C. J. (i96i), 'Trialkyltins and oxidative phosphorylation. The (3~p) phosphate adenosine triphosphate exchange reaction ', Biochem. 07., 79, 2 [4-219. BARNES,J. M., and STONER, H. B. (i958), ' Toxic properties of some dialkyl and trialkyl tin salts ', Br. aT. ind. Med., 15, I5-22. BLUM, M. S., and BOWF.R, F. A. ([957), ' T h e evaluation of triethyltin hydroxide and its esters as insecticides ', J. econ. EAt., 5o, 84-86. BLUM, M. S., and PRATT, J. J., jun. ([960), 'Relationship between structure and insecticidal activity of some organotin compounds ', ft. econ. EAt., 53, 445-448. CAVJOLLE, F., LESBR~, M., and MEYNIER, D. (I954) , ' Sur la toxicitd du tetramethylstannane et du tetraethylstannane ', C. r. hebd. Sdanc. Acad. Sci., Paris, 239, 556-558. DITTRmn, V. (I969), ' Chlorphenamidine negatively correlated with OP resistance in a strain of two-spotted spider mite ', .7. econ. EAt., 62, 44-47. FIs~E, C. H., and SUBBAROW, Y. (I925) , ' T h e colorimetric determination of phosphorus ', .7. biol. Chem., 66, 375-4oo. GARDINER, B. G., and POLLER, R. C. ([963), 'Insecticidal activity and structure of some organotin compounds ', Bull. ent. Res., 55, [ 7-2 I. GRAY, H. E. (I968), 'Plietran miticide--new approach to mite control ', Down to Earth, "3, 3-5HART[L, K. (I962), ' Triphenyl tin compounds ', Agric. vet. Chem., 3, [9-24. HUECK, H. J., and LUIJTEN, J. G. A. ([958), 'Organo-tin compounds as textile preservatives ', 07. Soc. Dyers Colourists, 74, 476-48o. KAARS SIJPESTEIJN, A., RIJKENS, F., LUIJTEN, J. G. A., and WILL~MSENS, L. C. ([962), ' On the antifungal and antibacterial activity of some trisubstituted organogermanium, organotin and organolead compounds ', Antonie van Leeuwenhoek, 28, 346-356. KERR, K. B., and WALDE, A. W. (I956), ' T h e anthelmintic activity of tetravalent tin compounds ', Expl. Parasit., 5, 56o-57 °.
1972, 3
ACTION OF TRICYCLOHEXYLHYDROXYTIN
LowRy, O. H., ROSEBROUGn,N. J., FARR, A. L., and RANDALL,R. J. (x95Q, ' Protein measurement with the Folin phenol reagent ', 07. biol. Chem., x93, 265-275. MmLER, G. L. (x959), ' Protein determination for large numbers of samples ', Analyt. Chem., 3 x, 964 • ProPER, G. R., and CASXDA,J. E. (1965), ' Housefly adenosine triphosphatases and their inhibition by insecticidal organotin compounds ', 07. econ. Ent., 58, 392-4oo. STONER, H. B., BARNES,J. M., and DuFf, J. I. (I955), 'Studies on the toxicity of alkyl tin compounds ', Br. 07. Pharmac., xo, I6-25.
I33
VAN DER KERK, G. J. M., and LUIJTEN, J. G. A. (1954) , 'Investigations on organo-tin compounds. III. The biocida|properties oforganotin compounds ', aT. appl. Chem., 'b 315-319Voss, G. ( 196 I), ' Ein neues Akarizid-Austestungsverfahren ftir Spinnmilben ', Anz. Schiidlingsk, 34, 76-77 •
Key Word Index: ATPase, Du-Ter, Plictran, tricyclohexylhydroxytin, triphenylhydroxytin, mouse, housefly, Musca domestica L., two-spotted spider mite, Tetranychus urticae Koch, spider mite ATPase, acaricides, organotin compounds.