University of Rhode Island University of Rhode Island

DigitalCommons@URI DigitalCommons@URI

Open Access Master's Theses

1968

An Investigation of the Relationship Between the Sulfhydryl An Investigation of the Relationship Between the Sulfhydryl

Reactivity and the Inhibition of Reactivity and the Inhibition of In Vitro Oxygen Uptake By Certain Oxygen Uptake By Certain

Molluscicidal B-Nitrostyrene Derivatives Molluscicidal B-Nitrostyrene Derivatives

Robert Thomas Louis-Ferdinand University of Rhode Island

Follow this and additional works at: https://digitalcommons.uri.edu/theses

Recommended Citation Recommended Citation Louis-Ferdinand, Robert Thomas, "An Investigation of the Relationship Between the Sulfhydryl Reactivity and the Inhibition of In Vitro Oxygen Uptake By Certain Molluscicidal B-Nitrostyrene Derivatives" (1968). Open Access Master's Theses. Paper 202. https://digitalcommons.uri.edu/theses/202

This Thesis is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons@etal.uri.edu.

~..ASTER OF SCIENCE THESIS

OF

ROBERT THO.MAS LOUIS-FERDINAND

Approved :

Dean of the Graduate School

l?~c~i~ UNIVERSITY OF RHODS ISU.ND

1968

AN Il\T\JESTIGATIOH OF THE R1:LATI01';SHIP

BE'IifSEN THE SULFiiYD':tYL RSACTIVITY

AND THE D!HIBITION OF IN VITRO

OXYGEN UPTAKE BY CERTAIN

MOLLUSCICIDAL Ji-~:ITROSTYRENE

DEIUVA TIVES

BY

ROBERT THOHAS LOUIS-FERDU:AND

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

PHA.RNACOLOGY

UNIVERSITY OF RHODE ISLAND

1968

ACKNOWLEl/GE!·IT::N1'S°"'--

'!11e author wishes to express his gratitude to his wife, Camille,

and his parents, Nr. and :Mrs. Joseph Louis-Ferdinand, for their

inspiration, generosity and understanding. The guidRnce provided by

Dr. George C. Fuller during the course of thes~ studies is acknowledged

and is sincerely appreciated.

'!11e zuthor also wishes to convey his thanks to his fellow students

for the many helpful suggestions offered during these studies. The

generosity of Dr. B. H. Pringle, who provided the snails used in this

investigation, is gratefully . acknowedged.

Financial support for this investigation was provided by the

U. S. Department of Interior, Office of Water Resources Research, under

P. L. 88-379.

ii

Louis-Ferdinand , Robert T. HS . University of Rhode Island, 1968. · An investigation of the relationship between the sulfhydryl reactivity and the in.~ibition of in vitro oxygen uptake by certain molluscicidal J3-nitrostyrene derive.tives. }:ajor Professor: Dr . George C. Fuller.

Eleven derivatives of p-nitrostyrene were four:d to inhibit the

oxygen uptake of whole tissue homogenates of the snail ~a1orbis

glabratus. The inhibition of oxygen uptake by ]3--nitrostyrene was

di~inished in the presence of excess and equimolar concentrations of

cysteine in the incubation media. The addition of cysteine resulted in

changes in the ultraviolet spectra of ]3-nitrostyrene solutions. Similar

addition of serine to ~-nitrostyrene solutions had no discernible effect

on the ultraviolet spectra . )3-nitrostyrenes were found to delay the

development of color which normally results from the reaction between

cysteine and 5,5 1-dithiobis (2-nitrobenzoic acid) (DTKB). The ability

of the nitrostyrenes to delay the progress of the reaction between cysteine

and DTNB was compared with the inhibitory effects of the same nitroolefins

on the oxygen uptake of homogenates . A positive correlation between -the

effects of the r.itroolefins on oxygen uptake and the effects on the

cysteine-IYT:rn reaction was found . These .clata indicate- that the sulfhydryl

reactivity of the compounds studied may be responsible for the molluscici-

dal activity of the nitrostyrene series studied.

i:U

1

TAS12 OY COHTE~:TS

Paee

ACJJ:d:!LSIXJENSNTS . • • • . ii

ABSTRACT . • • • • • • • • • • • •

TABIZ OF COI:'I'E!'ZTS • • • 1

LIST OF TABLES . • • • • • • • • • • •

LIST OF FIG mms • • • • • • • • • • • • 4

I. .DHRODUCTION • • • • • • • • •

II. SURVEY OF THE LITE...qATURE • • . , . . • • • • • • • 7

A. BASIS FOR SUL..H'HYDRYL RZACTIVITY • • • 7

B. DETEF.}1IllAllTS OF EOLLUSCICIDAL ACTIVITY. • • • • • • • • 7

c. HJ VITRO EVALUATION OF POTENTIAL PESTICIDES • • • • • • • 8

D. THE SEHSITIVITY OF BI03HERGETIC PROCESSES TO

SUL.'<'HYDRYL R3:AGE1,:TS • • • • • • • • • • • • • • • 9

III . EXP~L(IHEHTAL. • • • • • . . -. . • • • • • • • 11

A.. REAGEr!TS • • • • • • • • • • • • • • • • 11

B. • • • • • • .. • • • • • • • • • 11

c. ANALYTICAL PROC:ZDURSS . • • • • • • • • 11

1. Spectral Studies • • • • • • • • • 11

2. Sulfhycryl Reactivity. • • • • • • • • 12

J. In Vitro Oxygen Uptake • • • • • • • • . 13

4. Estination of Protein. • • • • . 13

D. STATISTICAI, EVALUATIOi.'I • • • • • • • • • • 14

IV • RESULTS • • • • • • • • • • • • . 16

A. EFFECT OF CYSTEDZ'.: 01! TEE ULT?..A. VIOL;:i;T SPZC'.l'Rlfrr

• • . 16

2

Paee

B. SUL..FHYDHYL ASSAY. • • • • • • 16

c. OXYGEN UFTAKE STUDIZS . • • • • • • • • • • • 28

v. DISCUS.SIOH. • • • • • • • • • 35

VI . SUIIHA .. "R.Y AND COIICLUSIONS . • • • • • • • • • • 38

VII. REFERENCES. • • • • • • • • • • • • • • • 39

Table

2

3

LIST OF TAB T.t<:S

Co!11parison of the effect of certain nitroolefins and phenyl­mercuric chloride on the development of colcr due to tho re­action between cysteine and 5,5'-dithiobis (2-nitrobenzoic acid) ........................................................ .

Effect of serine on the reaction between cysteine and 5,5'­dithiobis (2-nitrobcnzoic acid) in tl1e presence of )3-nitro-s~yrene •••• , ••••••••••••• .••••••.•......••••••••••••••• • • • • • •

Effect of certain nitroolefins en the rate of reaction betuee11 cy:>tein9 and 5, 5 1- d,i thiobis (2-ni trobenzoic acid) ••••

Page

25

26

27

Oxygen uptake of uhole snail hoinogena tes in th'3 presence of }3-nitrostyrene at J0°c ......•................................ 29

5 Oxygen upklrn of whole snail homogenates in the presence of f>-ni trostyrene and cystei."1e at 30°c... . . • • • . • • • • • . • • • • • • • . • • • 30

6 Effect of cert.a.in nitroolefins on the oxygen uptake of sna.il homogenates at 30°c .......................................... )2

7 Structural formulas of the nitroolefins used •••••••••••••••••. 33

3

LIST OF FIGUH.~S

Figure Page

1 Effect of cysteine on the ultraviolet spectra of p-ni trostyrene .•. .......••. ~ •..•..•..... .,.......... • . • . . . . . . . • • 17

2

4

5

6

7

Zff ect of serine on the u1 tr a violet spectra of ]3-ni trostyrene . ..................................•..•....••...

Effect of 5,5 1-tlithiobis (2-nitrobenzoic acid) concentration and et.~ylenedia~inetetraacctic acid on color stability of the product of the reaction bet·ween cysteir.o and 5 ,5 '-di thiobis (2-nitrobenzoic acid) •••••••••••••••••••••••••••••

Effect of ethylenediaminctetraacetic acid on the absorbance spectra of the product of the reaction between 5,5'-dithio­bis (2-P..itrobenzoic acid) and cysteine ••••••••••••••••••••••

Effect of time and ethylenedia.rai.~etetraacetic acid on the reactivity of cysteine with 5,5'-ditJliobis (2-nitrobenzoic acid) •• ••.....•.•..•.....••.•...............•....•..........

The effect of )3-nitrostyrene derivatives on color develop­ment due to the reaction of cysteine with 5,5'- dithiobis {2-nitrobenzoic acid.) . .•••.....••.•...•..•..•.•.•..•....••.•

The effect of 5,5 1-dithiobis (2-nitrobenzoic acid) concen­tration on the color development with cystei.Yle solutions in the presence of )3-ni trostyrene •.••••••••••••••••••••••••••

18

19

20

21

23

I. DiTRODUCTION

One approach in the worldwide e.tternpt to control schistoso:ne dor-,.

matitis has been erradication of the intermediate hosts through the use

of roolluscicines. Since several species of fresh 'I-rater snails have been

implicated as schistosone trans~itters in the western hemisphere (Cort,

1928) . a variety of substances have been proposed and evaluated for

molluscicidal activity. Thes19 include organo-metallic e.gents (Bond and

Nolan , 19.54; Frick and Jirninez, 1964), N-tritylmorpholine (Boyce, 1967) ,

sodium pentachlorophenate, and Bayluscide, (Sei ff er and Schoof , 1967) .

The paucity of evidence regarding the biological activity of these

agents justifi~s the pharmacological evaluation of these nolluscicides.

Research in this area would not only provide a theoretical basis for the

screening of potential molluscicides but would also suggest the clegree of

specificity to be expected from chemicals proposed for molluscicidal use .

Evidence published to date suggests at least two general classes of mollus­

cicides. Certain chemicals viz. disubstituted amides, are postulated to

act by a physical interaction with mer.ibrances or interfaces within the

snail (de Villiers and Rossom-1 , 1967). Others are believed to interfere

with enzymatic processes of the target ~rganism by interaction with ftui~­

tional groups of vital enzymes (Hack-worth, 1948).

The demonstration of the potent fungistatic properties of )3-nitro­

styrenes ·by Bocobo et al.,(19.54) suggested that an investigation of the

molluscicidal properties of )3-nitrostyrenes could be warranted. Inspec­

tion of the )3-nitrostyrene structure indicated that polarization of the

.t,-)3 double bond could provide a chemical basis for the known toxicity of

this group of compounds. It ·was felt that these conpounds would be

capable of reaction with org3nic thiol eroups and that such an interaction

might well provide a rational basis for an in y_?.tro screenine procedure

for similar compounds.

The studies to be described were desiened to determine: whether

nitrostyrene derivatives could be shown to inhibit vital enzymatic

processes; whether a suitable model could be found which would reflect

the ability of the test compounds to react with thiol groups of biolo­

gical substances and finally, whether any correlation could be demon­

strated between these properties.

6

II. SIBVEY OF }'.HE LITiq,ft.TURS

Basis for Sulfhydryl Reactivity

The potential biological toxicity of nitroethylenic substances was

indicated by Bousquet (1$43) who proposed that such substances might be

useful as pesticides. A chemical basis for the biological toxicity of

chemical warfare agents similar to nitroethylenic corJpounds, l·1as suegested

by Dixon and Veedham (1946). These authors attributed the polarization

of olefinic double bonds by adjacent electron-withdra•Ting groups to the

enhancement of the reactivity of such olefins toward nucleophillic re­

agents.

Nitroethylenic substances have been reported to react with sulf­

hydryl compounds in vitro. For example, Cason and Wanser (1951) were

able to react aromatic thiols with nitrostyrenes in the presence of cata­

lytic ~mounts of piperidine. Twait et al.,(1965) described the pyrolli­

dine-catalyzed. addition of nitrostyrenes and 2-a..'llinoethanol. Cavins and

Friedman (1968) studied the_reaction between acrylic acid derivatives and

protein sulfhydryl groups. In this study the authors presented evidence

which indicf!.ted that the acrylic acid derivatives were capable of reacting

with bovine serum albumin sulfhydryl groups at physiological pH.

Determinants of I·!olluscicidal Acthri tv

Evidence ha.s been published which indicates that certain non ole­

finic substances are also capable of altering ~ulfhydryl levels of exposed

snails. Devilliers (1963) reported that exposure of snails to nitrophen~

acyl chloride resulted in acid-soluble sulfhydryl levels which were 17%

of those of control unexposed snails. However, exposure to Payluscide

could not be shovm to alter acid-soluble su.lfhydryl levels. These results

7

were interpreted to suggest an enzyme-protectiva function for acid-

soluble thiols . Decreases in thiol levels were proposed to adversely

affect certain enzyme activities . Therefore the molluscicidal proper­

ties of Bayluscide could not be attributed to altered thiol levels

which suggested other determinants of molluscicidal activity.

...

Studies by Hackworth (19'-1-8) and Dixon (1946; 1948) indicated that

enzymatic inhibition resulting from exposure to certain chemical warfare

agents and molluscicides , could be mitigated in the presence of cysteine

and gluta.thione . These data ·were taken to suggest that the toxicity of

these agents was due to the inhibition of vital enzyme systems which

required intact sulfhydryl groups for normal activity. However deVilliers

(1967) found that maximum snail lethality could be obtained when snails

were exposed to disubstituted amides with substituent acyl chain lengths

of 12-13 carbon atoms . In addition , exposed moribund snails could be

revived after repeated washing. Devilliers concluded that physical

interactions of the molluscicides with snail membranes played a dominant

role in the molluscicidal activity of these compounds .

Von Brand (1S49) observed that~-nitrostilbenes inhibited 99-'P of the

oxygen uptake of Australorbis glabratus . Overnight washing of the

exposed snails in aerated water did not return the oxygen uptake to

levels greater than J8% of the pre-exposure values . The absence of

recovery indicated some differences between the reversibility of the

effects of the disubsti tuted amides and ..t-nitrostilbene.

In Vitro Evaluation 2f Potential Pesticides

Yuki et !tl• ,(1966) atte:npted to relate the chemical reactivity of

antiviral agents to the virulence observed by comparing thiol levols

before and after incubation of the compounds with methyl N-acetyl

8

cysteinate (EAC) . It was found that compounds which were capable Of

inactivat:ing Adenovirus type 5, were also capable of reacting with

sulfhydryl groups of 1-L\C. Inactive com.pounds did not react with HAC.

Ho11ever , all MAC-reactive substances did not possess antiviral properties .

Nabih and Elwasimi (1968) determined the influence of Bayluscide

on the biotransf ormution of solutions of N-N-d:i.methyl-p-phenylenedia.rnine

or hydrogen peroxide by intact snails . The reduction of color developnent

or peroxide decomposition in t..h.e presence of the molluscicide was used

to measure interference with cat.a.lase or peroxidase activity.

Tweit et al. ,(1965) found that while,8-nitrostyrenes were potent

antibacterial n.gE>nts :in vit~~ the in vivo activities were less t..1-ia.n ex-

pected. This discrepa.ncy Has attributed to the irreversible binding of

the )3-nitrostyrene to thiol or amine groups of blood . These results uere

in accord with those of Evans et al., (1956) in which preincubation of

fungi with )3-nitrostyrene was found to protect mice . However , administra-

tion of )3-nitrostyrenes afforded little protection to mice which had been

pretreated w1.th.fungi.

The influence of }3-nitrostyrene derivatives on oxidative phosphory-

lation in rat liver mitochondria was studied by Bovell et al., (1964) . j3-

Nitrostyrene derivatives i·rere found to uncouple oxidative phosphorylation

and inhibit energy-dependent processes . However , at hi~er concentrations

these agents were found capable of inhibiting respiration.

~ §ensitivitz of Bioenergetic Processes to Sulfhydryl Reagents.

Evidence has accumulated which suggests that bioenergetic processes

are susceptible to sulfhydrj·l-reactive agents . Conceivably, oxidation of

free protein groups on the sa_'11o or on different peptide chains could result

in alteration of the original protein structure (Hest et al. (1966) . __ ,

9

Accordingly enzyrie activity and/or membrane ·properties could be affected.

Penefs°k'J (1967 ) has shcr-1m tha.t decreases in the detectable sulfhydryl

group content accompanies loss of ATPase activity. Ia.~ (1968) has

indicated that dithiols can reduce the extent of reagent-induced or

storage-induced loss of energy-linked P'Jridine nucleotide reductase

activity. Dithiols have also been shown to reverse arsenophonyl­

associated inhibition of oxidative phosphorylation (Fluharty and Sanadi ,

196J) . Iodoacet!lmide and p-chloromercuribenzoate have been shown to

inhibit the respiration of tissue minces prepared fro::n .Austra.lorbis gla­

bcatus (:·J"einbach , (195J) . These findings have been i.l'lterpreted by the

autJ.1or to su.geest the presence of sulfhydryl enzyme systems .

Thus the vulnerability of living systems to sul:fhydryl-reactive

substances is well established. The reactivity of certain..£;8 unsaturated

compounds ·nith protein sulfhydryl groups indicates that the nitrostyrenes

may exert their toxic effects by interaction with biological systems which

are susceptible to thiol-reactive agents . This study has been designed

to determine t..~e ability of certain nitroolefins to interfere with the

reaction be"b.1een cysteine and 5,5 1-dithiobis (2-nitrobenzoic acid) and

to determine the extent of correlation with the ability of the sar.ie nitro­

olefins to inhibit oxygen uptake .

10

11

III. EXPERU~NTAL

Re<Jgents

Reagents used were anzlytical grade or equivalent. Cofactors, sub-

strates and amino acids used were obtained from Calbiochem of Los Angeles,

California. 5,5'-Dithiobis (2-nitrobenzoic acid)(DTNB) was used as ob-

tained from Aldrich Chemicals of :Milwaukee, Wisconsin. The molluscicidal

agents were prepared by the Pharmaceutical Chemistry department at the

University of Rhode Island.

Animals

Laboratory reared Australorbis glabratus were provided by Dr. B. H.

Pringle of the Northeast :Marine Health Sciences Laboratories, Narragansett,

R. I. Once obtained, the snails were maintained on an ad libitllm diet

' of lettuce in glass beakers which contained 65.3 mg sodium thiosulfate

pentahydrate per liter of tap water.

Analytical Procedures:

. Spectral Studies

The effects of two amino acids (cysteine and serine) on the ultra-

violet spectra of B-nitrostyrene solutions (O.luN/rnl in O.lM phosphate

buffer pH7. 3) were determined. After blanking the_ instrument with

phosphate buffer, the 1-cm cuvettes which contained 2 ml of the B-nitro-

styrene solution were placed in a 30°c thermostat-controlled chamber of

the Beckman DBG spectrophotometer. Once the initial B-nitrostyrene spec-

trum had been recorded, successive 0.1 ml aliquots of luM/ml solutions of

the amino acid were added to the sample and reference cuvettes. A

final addition of amino acid (0.1 ml of a 0.02rnN/ml solution) was used.

The resulting spectrum was recorded following each addition for comparison

with the i'1i tfal J>nH.rosty.i:·one spectrum•.

Sulfhydryl reactivity was deterrD.ned b:r follo~-ling the rate of the

color-fornil'.,J reaction between cysteine· and 5,5'- dithiobis (2-nitrobenzoic

acid) (DTNB) in the presence and absence of the suspected molluscicidal

agent. The incubation media contained 7 ml cystBine (0.25Il".H), 10 ml

et.hylenediruninctetraaceta.te (EDTA) (0.5i'l) ad.justed to p!i7 .3 with

pote.ssirnn hydroxide, 5nl deionized water and 6 ml (pH?. 3) 0. lH phos-

phato buffer. Successive additions of solutions of t.1-ie nitroolefins in

ethanol were :i.ncubated ·with the test miitures. F.q~l volumes oi' ethanol

-were added to controls and the mixtures were incubated in a Dubnoff

incu1:a tor f.or 20 minutes at 30° c. 'l\.ro r.i1 aliquots were U1en pipetted

into faro lrn litOit path spectrophotometer cuvettes which in turn were

placed in a 30°c thermos-tat-controlled cuvette chamber of a Bed:nan DP.G

spectrophotometer. Ttrnnty-five microliters of DTNB (4me/IT'J.) were pre-

pared fresh daily in O.lH phosphate buffer pH7.J and were pipetted :into

-the sa.~ple cuvette to start the color developnent. Five seconds later

the develop;nent of color was monitored by measurement of the absorbance

at 412riu. An accessory Beckman 10 inch l:Lncar-log recorder was used in

these studiss ~ The pH of the fi11..al incubation mediun was r:ieasm·ed at

the end of the assay period. The rate of a.bsorbance increase was measured

fro;11 straight line portions of the recordings. Values used were the

arithmetic means of at least bro deterrninations. Tne reciprocal slopes

calculated fro:n the spectrophotonetric determinations were taken to

represent sulfhydryl reactivity. The addition of IYI'NB to aliquots

renoved fro:::n a similar incubation Medium fror.i "iihich cysteine had been

excluded resulted in negligible absorption at li.12zm.

12

lJ

-- --- --- ---Ox:nen up~ke was m<:.asur.~d r..anometric~lly. V olu:-:10s of homo~ena tes

rr.nged frc.n 1.0~-0.2 n-J.. Pro:incuL""12.tion was alloued to proceed at )0° C

for twenty mi.'1utes in 16-JDcc 1-rarburg flasks 11hich cont-:i.h1od the incuba-

ti on neclirrn unless othertrlse noted.. Incubation medilll-:i 1A1 was usod in

init:L'.11 studies and contained (i..'1 a fir.3.l voltu!le of J.2 rnl) 6 iinoles

po-W.ssiuin i'u.'llr:i.ra te, 6 u,~1oles potassiu.11 pyruva te, 12 urrioles rn.agnesi'UI11

chloride, .3 umoles adenosine triphosphate, 585 u.:1oles s~crose and O.Jrnl

(pH 7.3) b.lH p'hosphate buffor. Sufficient distilled water was included.

to attain t..lie desired final volmno follouing the addition of the homo-

genate. Incubation medium 'B' contained (in a final volume of 2.?ml)

5 µmoles ci.denosine 5' diphosphate, 12.5 \L!loles succinic acid, 1 • .3 umoles

magnesiu.11 sulfate, 625 urnoles sucrose, 0.375 ml of o.nr p...1-iosphate p[I

7.) buffer e..r.d O.lJ. !i"J. Of hOBOgenate,

Solvents used ·Hel"e eith9r absolute ethanol or water-methanol 91:9

(v:v). An equal volume of the corresponding solvent Has added to the

final control incubation medii,w.. Two tenths ml of 20p aqueous potassiu'1l

hydro;:ic!e uas added to the center well of each flask. The cofactor

solution ~·1as tipped in from a side arm at t."ie commencement of tJ1e measure-

ment period. Follmd.ng t..l1e measurement of the ox-.fgen uptake in the

presence of the nitroolefins, the contents of a group of flasks were

pooled and the pH ·was measured. Aliquots of boilf.ld hoaogenates were

incubated with both media in the presence of nitroolcfin and the apJX!.rent

o:xygen uptake ne?..sttred were neglic;ible. All readings were corrected for

changes in baro:;:.0tric press11.re throueh the use of a thernobaronieter.

Estination of Pro'kin

The protein content of homogenates was estinated by the color:inietric

. procedure of Lo-:·ITY et £!..I (1951) ui th r.tinor modifications a A 0. 2itl

aliquot of homogenate was adci.ed to 15ml test tubes containing 10 ml of

lN ·sodi~ hydroxide, im;tead of pot?.ssium hyr.1roxide . The use of sodium

hydroxide eliminated the formation of precipitates in the fi.n.11 solution.

The tubes were heated in a i·1ater bath until dissolution was CO!nplete .

Stanclar<l solut1.ons of cystalline bovine serll':l alhur.d.n 2.nd a blank

consisting of 0.25H sucrose were carried through the entire procedure .

When the tissue was completely dissolved a 0.2 ml aliquot was removed

from each tube and was placed into a second tube containing 1 ml of 0.5N

sodimn hydrcrAide solution. Five ml of reagent 'A' (prepared by the addi­

tion of 1.0 r.i.1. of 1% cupric sulfate solution plus 1.0 ml of 2.7% potassium

ta.rtrate , to 100 ml of .2p sodium carbonate solution) was added to ea.ch

tube • mixed and t..Yie tubes allowed to stand at room temperature for tuenty

minutes . At this time 0 ~5 ml of reagent 'B' (prepared by diluting . .

commercial Folin-phenol reagent with an equal volume of distilled uater )

·was added to each sample with rapid mixine . The sa..r:1ples were allowed to

stand at roo:n tenperatm·e for L~O minutes for color development. The

absorbance ·of the solutions was determined against the blank in a model

DEG Bockman spectrophotometer .

Statistical Evaluation

The Student's 't' statistic(Fryer, 1966 ) uas used to determine

whether statistical differences existed 'between means .

The equa.tion used W<?.s : t = x1-x2 6 il/N1 +171!2

where 6

: H1 si + N2S~

Xi and x2 == sample means

Nl and N2 == sample sizes

2 s2 sample variances s1

a.nd == 2

14

The calculated 1 t 1 statistic "Has then compared with critical values

obtained from the 1 t 1 distribution tables. Hypotheses of identity wero

rejected if the calcula.ted statistic exceeded the bounds of t..~e critical

values. (p<0.05)

Spcarman 1s formula. (Spiegel, 1961) was used. to detemine salllple

correlation coefficients. Nitroolefins were ranked according to potency

as molluscicides, :inhibitors of O".-<ygen uptake and sulfhydryl reactivity.

The eqU2.tion used was:

r = sample coefficient of rank correlation

r = 1 - 6 !D2 f.. D2 = sTu11 of the squares of the cliff erences

nC11-1) between ranks of compared vales

N =number of pairs of values.

The 't' statistic was used to test the hypothesis that the population

coefficient of correlation, denoted by J was equal to zero. The equation

used.was:

r = sample coefficient of rank correlation

t=rfN=Z ~__.l-r2

N = number of pairs of values co~pared

t = 't' statistic

The calculated 1 t 1 statistic was then compared with critical values for

the 1 t 1 distribution. HypotJieses of equality behreenf and zero .were

rejected if the calculated statistic fell outside the bounds of the

critical values (p< 0.05).

15

IV . RESULTS

The effect of the addition of cysteine solutions on the ultra-

violet spectru.,11 of )3-nitrostyi·er.e is sho:m in Figure 1. The alterations

observed include a decrease in the absorption at JOO mu, an i.~creasa . in

t.~e absorption at 250 mu and a progressive shift in the absorption maxi-

mum at 230 mu to shorter wavelengths . Isosbestic points in the family of

spectra can be seen near 263 mu and 250 mu. The results of similar ·

additions of serine to ~-nitrostyrene solutions are presented in Figure

2. No qualitative spectrum changes are discernible . The quantitative

alterations seen are no greater than those to be expected from dilution

of )3-ni trostyrene solutions . The absorbance of }3-nitrostyrene follows

Beer 1 s law through a concentration range of 0. 0 5 to 0 .15 pl·~/rnl .

Sulfhydryl Assay_

The effect of ethylenediaminetetraacetate {EDTA) on the stability of

color formed following -the addition of different concentrations of DTNB . to cysteine solutions is shown in Figure J. The decrease in absorbance

proceeds at the same rato in the presence of either 0.025mHolar or 0.084

mHolar DTNB. The use ·of 0.08 molar "SDTA results in color stability for

at least JO minutes . Comparison of the absorbance spectra obtained with

and without EDTA {Figure 4) indicates that EDTA docs not alter the absor-

bance spectru.'11 of the color formed following the reaction between cysteine

and DTNB.

The presence of EDTA was also found to E-nhance the stability of cy­

steine solutions . {Figure 5). The estimated half time for reactivity of

cysteine with DTl!B in tho absence of EDTA ranged from 8 to 4 minutes .

16

w 0 z < IJ.'.l lli 0 U)

IJ.'.l ·..i;

F"IG-. I.

0.70

0.60

0. 50

0.40

0.30

0.20

o. 10

,, .. -·•\

' \ . \ •

EFFECT OF CYSTEINE ON THE ULTRAVIOLET SPECTRA OF,8-NITROSTYRENE

• , ¢ , I

, • ~ ,. ' I •

C!> '/!. ' 1 • , ' ' ' ·~' '

I I ' ' '4

, ' I\ \ • I

' ' \ \

' I

' \ ' I • ' ' \ ' ,

I \ ~© (!) I q \ ' I

' ' ' ' ,

I \ ' , ~ " I \\ • I \ 0

I

' I

' t • "' ' • I I \.,\ ' . / p 9

\

' \ \

' \

Ql t

\\ 0 I

I

'\ ' t '"" ' \ ," 0 I I

, ' I

p' \~ \ ~ , I ' ,. \ I ,

I "., · -~ J5,. ', """-0' ,---' I - " ,•

\ ............... ...[)' \

\

\

() '\

\

\

\

ca

I ~

I I

n I

I

' 0 ......

230 240 250 260 270 280 300 Wave Length {rnµ)

(0-0) .8-Nitrostyrene 0. 1 )lm/ml; (L\"'-L\-) 0. 10 pmole cysteine plus f-}-nitrostyrene; (0-0-) 0. 20 prnole cysteine plus f->-nitrostyrene; {-0- o-) 2. 2 Jlmole cysteine plus)3--nitrostyrene

17

..

Fl"G • 2. EFFECT. OF S:::Ril.JE OH THE .ULTRAVIOIEr SPECTRA OF ]3-HITitOSTYRENE

0.70

0.60

0.50

o.4o

~ (.)

i 0.30 ti)

ea

0.20

·0.10

,, I I

1, I

~-, I I I . \ I

I ' ' .\ I I I

,,. -,\ /I I

\ , , , _ ... , \ I f

I '\ \ f' I

'\ I ' I f I I

' f 1 I

' I ' '

\ I 'I I II I I c

' \ I It ,,

1 , , ' , ,

' l I 11 ., I I 1

l ' •, I 1

1 I I ' - \ I /I

\ 1, 1 I

I J I

\k I I I ~ , I I

\ .:-- ~ '_,,,, I ,,_ -'

240 250 200 270 200 zJo)-do--1 Have length (r.i.µ)

A

B

c

A: )3-11itrostyrene 0.1 p:r.i/rtl.. ; Jl: A plus 0.1 >trnole serine ;

C: A plus 2.2 )t!lOles serine .

18

~ u z za ~ ~ <

FIG. J. EFFECT OF 5,5LDITHIODIS (2-t:ITf\03S!nOIC ACID) CO!:CS!iTHJ.Tl(;N AND ETHYLSl'EDIM!I.NE'rSTRAACE'?IC ACID ON COLOR STltBILITY OF THS PRODUCT o~· THE REACTION B~.l.1'EEil CYSTEI! '. E A~D 5, 5 '­DITHIOBIS (2 -NITRODEt;zorc ACID)

0.9

o.8

o.6

0.5

o.4

O~J

0.2

0.1

--o----- -o- -

-0

v-o_ -... o ... o ... - - .......

~- " ..... ...... 0-. ...... ....... o ....... v-

___ _:.._ ___ .IL.---~~·L ______ -· ~'~--10 20 JO ·- 40

TDlE (min.')

(·0··01 0.08 ml·:olar DTKB plus 0.083 molar EDTA f-Q··Q~ 0.025 ml:Olar DTNB; f\j·-V·) 0.08 ml1olar DTNB

19

r:cl u

~ 0 Cl)

~

FIG . 4. EFFECT OF ETIIYLSHEDIA11IHETETRAACETIC ACID ON TH:<: ADSC?J.3.\ECB SPECTP.A OF TH8 PRODUCT OF T.tIE 11~CTION B2TL·TEEN

5,5 1-DITHIOBIS (2-NIT.ROBEHZOIC ACID) AllD CYSTEDlE

1.0

0.9

o.8

0.7

o .. 6

0.5

o.4

O.J

0.2

0.1

, o-o., , . ,. D

tl \ With ED'rA I \~~~~~~~-

I

I

J 0

I J

I ,

I

0 " \ o'' 'o \

\ \ ., ' . o \ ' \ \

\ \

' . Q\ ,_, ____ _..Without EDTA

\ \ \ \

\ a \ \

o' \\

\

' ' ' : 0 'o' '" ,,

"\ ' ... -,'.380 400 420 41-~0 460 430 500 520

WAVELENGTH (m)l)

20

FIG. 5. EFFECT OF TIME AND ETHYIEl·~DIAHTI.ffiTETRAACETIC ACID on THE REACTIVITY OF CYS'IZmE WITH 5,5 1-DITHIODIS (2.-lHTROBDiZOic· ACID) w;:

1.0

o.8

o.6

-- v-o- \/-' 0

0- 0 '1- - '1-

\ \ \.

\ ' \ '

\ \

~ \

\ \

\ ' \ ' \ \

\ \

'\

t o, \

\

p\ \.

\ o .... \ '

5 10 15 20

TINE STANDJJIG (HIN.)

(-a-a-) 75 J.1."1.0lar Cysteine pH8.0 at Roor.i Temperature (·0-0-) 75 pmolar Cysteina pH7. 0 at Room Temperature (-V-\7-) 75 ):t..'llolar Cysteine pH8.2 , 0.08rr.olar EDTA at Ro8:n Temperature (-()-0-) 63 pmolar Cysteine pH7.J, 0.15molar EDTA at JO c.

21

In the presenco of O. 08 1·1olar EDTA, the absor'oo.nce obtained on the ndc1i­

tion of DTim to a cysteine solution which fl.ad been kept at roo1n temperature

for 15 minutes in pH 8.2 phosphate buffer was 88~ of the extrapol<l.ted

zero time value. It was estimated fro!n the decay plot that the absorbance

at 412 mu for cysteine solutions (incubated for 15 minutes at J0°C in

20ml·:ol~r pH 7.3 phosphate buffer) in tho presence of 0.18 Eolar EDTA was

<JJf, of the initial values.

The absorbance obtained following the reaction of DTHB with cysteine

in the presence of 0~18l·~olar EDTA was found to be linearly related to

concentration in the concentration range used. The absorbance of a 1

molar cysteine solution in a 1 cm CU'Jette under the conditions of this

assay was determined to be 13,400 absorba...n.ce units.

The development of absorption at 412 mu, folloning the addition of

DTNB to a solution of cysteine is shown in Figure 6. The absorption

approaches a maximum value within 0.03 minutes after coriunenceI'!ent of the

measurement period. In the presence of 0.13 rnHolar J>-nitrostyrene, the

absorbance increases more gradually. An excess of DTNB does not increase

the rate at which the absorbance changes in the presence of J ·-nitrostyrene

(Fieure ?). v!hen cysteine is o~itted from the reaction medium, the

addition of DTNB results in an absorption of 0.015 absorbance units with

0.26m!·f }3-nitrostyrene i.Yl the incubation Medium with no further change in

the ·absorbance. The interference •-rith the progress of the rea.ction increa­

ses with increasing p-nitrostyrene concentration. In t..~e presence of

an excess of cysteine, viz. Jmi·:olar, the effect of-)3-nitrostyrene concen­

trations up to O.h9rnHolar are not discernible.

The effect of certain nitroolefin derivatives and phenylr.:crcuric

chloride on the reactivity of cysteine solutions with DTNB is shmm in

22

FIG.6

o.s

0.7

N 04 r-1 • -::I

E--< < ('\ ,, r-c.l Vo)

0 ._,.. fl"·-!

<:'!

SQ 0.2 8 I ~ ~ 0.1 !

THE EFFECT OF .B-NITROSTYRENE DERIVATIVES ON COLOR DEVELOP1'1ENT DUE TO THE REACTION OF CYSTEINE WITH 5,5'-DITHIOBIS(2-NITROBENZOIC)ACID

. URI-237

0 .01 0. 05 0 .10 0.10 0.20 0 .30 o. 4o 0. 5c

o" (

I 0

I

'/ .D I

I

y

0 ... o

,.o -o .. o·o·D­

..o'o

URI-4

l . {j ?.. • (~ .) e \ J ' i• • { .' .) • ' ' J • I I

TIME(min.) C-.1steine concoOo075mMola.r;URI-2J7 cone. {00)0.24nu'1ola.r; {6-A-) 0.5Jw'1olar;URI-4 cone. (0-QJO.lJ~lolar E'v-7-)0~J6rr.I1olar; P-0-)Ethanol0.2rrJ./ml reaction medium.

I\)

1v1

FIG . 7. Th.:!; SFFECT OF 5, 5 '-DITHI08IS(2-NIT~{03.&'l.!ZOIC ACID) CONCENTRATION Ol~ COLOR. D:-i:VELOPM&'JT WITH CYSTEINE SOLUTIONS IN THE PRESENCE 01'' }~-NIT RO STYRENE

(/) ;-~

g 0 H -~ H .....:1 .....:1 rl ~

N rl .::::[

E-< ~

r_,,~ 0 z ~ (.:'.l ()'::! 0 Cf) cc <I!

o.s l 0. 7

o . 6

0. 5

o.4 J O. J I

0 . 2

0.1

/ 0

0J5

0,,,,,

...-0,,,,,. ,o

TIEE(min.)

,..,,,. 0 --o 0 ...... -

C-;steine cone. 0 . 07 5m..11olar;)3-Ni trostyrene cone. 0. lJmHolar; 5, 5' -Dithioois ( 2-ni trooenzoic acid) cone .; (0-0)0 . 028rm\Jolar; (00)0 . 084rrJfolar ; (A-1:..· )O . JJmMolar; (O-D)o . 66mi'1olar.

TABLE 1

CONPARISON OF THE EFFECT OF CERTAIN NITROOLEFINS AND PHENYL~IBRCURIC CHLORIDE ON THE DEVELOPMENT OF COLOR -DUE TO THE REACTION BETWEEN CYSTEINE AND 5,5-DITHIOBIS (2-NITROBENZOIC ACID)

AGENT

URI-110 0

JJ H H CH3CHzc-o-Q-e= C-N02

URI - 4

H H

Q-c = C-N02

Phenylmercuric Chloride

CONCENTRATION PPM mNolar

30 o. 13

98 0.43

26 0.17

67 0.42

137 0.88

3. 6 0.011

I/Slope(min./OD)

10.l

17. 9

12.3

23.8

45.5

143.0

Cysteine 0.017 mNolar . Phosphate pH 7.3 buffer, 0.026 M; nitro­olefins added in absolute ethanol. Phenylmercuric chloride added in deionized water. 5, 5-dithiobis (2-ni trobenzoic acid) 0. 084 m1'1olar.

25

TABLE 2

U•'F',~CT OF SERINE ON THE REACTION BE'IHEEN CYSTEINE AND 5,5 1-DITHIOBIS

(2-NITROBENZOIC ACID) IlI THE PRESENCE OF ~-llI1'ROSTYRENE

REACTION SYST.211 HEAN SLOPE (OD/min) l

---~~----------~-------..,._~-~------------~------------------------------Cysteine ( 0. 086 mI~olar)

)3-Nitrostyrene (O. 08~ mI:!olar)

Ihosphate buffer pH 7.3 (0.026 Eolar)

ED'i'A (0.22 Holar) 0.360 -----------~-~-------------~------_ ...... ___ ...___ __________________ ~---------Cysteine (0.086 mHolar)

fr-Ni trostyrene ( 0. 08.3 m.r-~olar)

Serine (0.086 roHolar)

Phosphate buff er pl 7 • .3 ( 0. 026 }folar)

EDTA (0.22 Eolar) 0.366 -------------------------------------------------------------------------. '

Serine (0.086 mlfolar)

}3-Nitrostyrene (0.08) mHolar)

Fhosphate buffer pH 7 • .3. (0. 026 Molar)

EDTA (0.22 Molar)

1values represent the mean of duplicate assays.

2

20n the addition of DTNB, the absorbance increased to 0.02 ·with no

fUrther ch2.nge.

26

27

TABLE J

EFFECT OF CERTAIN NITROOIEFINS ON THE RATi!: OF REACTION BE'IHEEN CYSTETI-JE

AND 5, 51- DIT.dIOBIS (2-NI1'ROBEHZOIC ACID)

AGENT1 NITROOIEFJJI FLASK CONCENTRATION l/SLCPE (min/OD)2

URI-4 . 0.25 mHolar J.46

URI-5 0.25 mHolar 3.61

URI-7 0.21 mHolar J.18

URI-8 0.26 mMolar 1.66

URI-97 0.27 mJ'folar 1.19

URI-99 0.27 mHolar 5.56

URI-119 0.24 mHolar 2.46

URI-120 0.20 illI1olar 2.54

URI-129 0.21 mMolar 5.24

URI-lJO 0.27 mHolar J.50

URI-237 0.22 mMola.r ___ 3

1 Structural formulas of nitroolefins are presented in Table 7.

~ 2v~lues represent the moan of duplicate assays.

Jl•Jaximum absorb8.nce was reached within ten seconds.

28

Table 1. At this concentration of cysteine (0.017mHolar) the initial

slope of the absorbance-time recordine decreases 'With increasing )3-nitro-

styrene concentration. The presence of J.6 parts per million phenyl-

mercuric chloride results in a. greater degree of interference with the

developnent of absoroonce at 412 mu than would be obtained with )3-n:ltro-

styrene. To simplify comparison of subs~~nces according to the ability

to delay the progress of the DTNB-cysteine reaction, the reciprocal slope

has been used • . From Table 1 it is seen that the reciprocal slope in the

presence of phenylmercuric chloride is approximately 10 times greater than

that in the presence of J3-nitrostyrene. Saturation of the..(-p double bond

of )3-nitrostyrene results in a product in which the ability to interfere

with the DTNB-cysteine reaction is considerably diminished (Figure 6) .

The interference by )3-nitrostyrene with the sulfhydryl assay was

' determined in the presence and absence of serine in order to estimate the

specificity of ~-nitrostyrene for thiol compounds under the conditions of

this assay. From Table 2 it can be seen that the rate of color develop- ·

ment resulting from the reaction between cysteine and DTNB was essentially

the same in the presence or absence of serine. The effect of the nitro-

olefins on the sulfhydryl assay is sho~m in Table J. The concentrations

of nitroolefins used in oxygen uptake studies were 1.47 times greater than

the concentrations used in the sulfhydryl assay.

Oxy~en Uptake Studies

The uptake of oxygen by homogenates (incubated in medium 1A1 ) in the

presence of ~-nitrostyrene decreases with increase in nitroolefin con-

centration (Table 4) • The concentration of ,B-nitrostyrene at which the 4 .

oxygen uptake approached 50p of control levels was determined from inspec-

tion of the dose-response plot to be o.41} mg~. J3-nitrostyrene was found

TABLE 4

OXYGEN UPTAKE OF WHOLE SNAIL l HOEOOENATES DI TIIB · PRESE!!GE OF }-

NITROSTYRENE AT 30°c

CONG. lUTROSTYRSl;E

rog% mHolar

------0.17 0.011

o.Y.,. 0.023

o.84 0.056

1.12 0.075

1.68 0.113

CONG. :NITROSTYRENE

,d . mg}> rnl·rolar

2.4 0.15

4.8 0.30

9.6 0.60

19.Z 1.20 1Australorbis glabratus

MEDIUl{ 1A1

60 1-:Tln.JTZ 9z UPTAKE

u1

44.o

41.0

25.1

10.7

3.6

7.5

UEDIUH I B'

~ HINUTE 9z UPTAKS

ul

36.9

28.3

25.4

16.9

10.5

! CONTROL

100

9).2

57.1

24.4

8.2

17.0

1'_ CONTROL

100

76.7

68.8

45.8

28.5

2i.IediUlll 'A' contained in a final volume of J.2 ml per warburg flask: 6 umoles potassiu.~ fumarate, 6 u:~oles potassiu:n pyruvate, 12 u::ioles l·:gCl2 , 3 umoles ATP, 585 umole::; sucrose, 0.3 ml O.l J.:olar phosphate bUffer pH 7.3 and 0.6 ml of 50 ~{,whole snail homogenate, containing 8.4 rng/ml protein. !,foditL."n 1 B1 contained (in a fil"..al volu:ne of 2. 7 ml) 5 umoles ADP, 12.5 umoles succinic acid, 1.3 u:--:oles HgS04 , 625 umoles sucrose, 0.375 ml O.lH phosphate (pH 7.J) buffer and 0.4 ml of homogenate (29.2 mg/ml Protein).

29

'i'ABI,E 5

OXYGEN UPTAKEl OF \·!HOLE SliAIL 2 HOi·1CGENA TZS IH THE PRZSEI~CE OF p-

NITROSTYRENE AED CYSTEINE

FLASK COl!CENTRATION FORTY MINUTE ~UPTAKE <!?_ CONTROL

llITROSTYREHE CY STEINE

(mg~~) (mHolar) (mr·rolJJr) (ul)

------- -------- 6 .,. 4 3 .9-1.7 100

------ 1.2 39.Bt 1. 94 108

4.8 0.3 .1.2 30.l! 1.9 81.6

19.2 1.2 1.2 21. 7±1.2 58.8

19.2 1.2 10.5:!' O.J 28.5

1rncu.ootion medium 1 B1 was us~d in t1'.is study. The mediu.'ril contained

(in a final volUJ~e of 2.7 ml) 5 untoles ADP, 12.5 urnoles succinic acid,

1.3 umoles Hgso4, 625 umoles sucrose, 0.375 ml 0.1 Eola.r phosphate pH

7 .3 buffer and. 0.4ml of whole s!'..a.il homogenate. Protein concentration

was 29.2 mg protein/nll. homogenate.

2 · Australorbis glabratus

3vaiues shc;,m are the means:! standard er:r·or for groups of at least three

determinations.

4statistically identical (p (. 0. 05)

JO

to affect the uptake of oxygen by homogena.tes incubated in medium 1B1

to a lesser dos;ree . Tho flask concentration at which tho oxygen uptake

was .50? of control values was dcterrdnsd to be approximately 16 ti'T!es

that concentration of )3·nitrostyrene producing the same degree of inhi­

bition whon tissue was incubated in medium 1A1 , viz . 7.&ng~ (Table 4).

In view of the reduced levels of o:x7gen uptake reported by Weinbach

(19.53 ) when pyruvate was used in sh"'lilan studies, medium 1B1 was used

in subsequent studies.

When cysteine was e..dded to the incubation medium prior to the addi­

tion of )3-nitrostyrene , the levels of oxygen uptake were greater than tha.t

of homogenates in the presence of ~-nitrostyrene alone . Tables 4 and 5

shm1 tha.t incubation of equimolar concentrations of cysteine and)3-nitro­

styrene resulted in mean oxygen levels which were approxirlately ih·lice

those of an aliquot of the sa..me preparation in the presence of )-nitro­

styrene alone . Accordingly the mean oxygen uptake of homogenates in the

presence of an excess of cysteine Has 10'.h greater than .t'hat of homogenates

exposed to the sa..:-ne nitroolefin concentration. The mean Oxygen uptake in

the presence of cysteine alone was not statistically (p<0.05) different

from controls .

Inhibition of snail homogenate oxygen uptake was observed at nitro­

olefin concentrations ranging from O.JO rr£.foles/ml to 0.44mEoles ml

(Table 6).TI1e oxygen uptake determined in the presence of the ft-nitro­

styrene derivatives was 46 to 80 percent of control values . The mean

pH of pooled control media (:r:ieasured at the termination of oxygen uptake

determinations) was 7 .3 ! 0.2 while that of media. which contained nitro­

olofins ·was 7 .4 .!: 0.01 . Rankine of the nitroolefins a.ccorcling to the

ability to inhibit t.~e color development of the sulfhydryl assay, and

31

TABLE 6

EFFECT OF CERTAill liITROOLEFn;s ON THE OXYGEN UPTAKE1 OF SNAIL 2

BOl·IOGENATES

AGEl\ir3 FlASK CONCEHTRATION ( rnM) FORTY MINUTE o2 UPTAKE4

URI-4 0.37 21.0 = 2.0

URI-5 0.37 17.7: o.6

URI-7 0.30 23.5 ~ 4.4

URI-8 0.38 + 29.1- 0.3

URI-97 o.4o 19.2 :t o.6

URI-99 0.39 16.8'.tl.6

URI-119 0.36 27.1 ± 2.5

URI-120 0.30 18.0 :!: 1.2

URI-129 0.32 + 21.0- 0.9

URI-130 o.4o 18.9.!2.l

URI-237 0.33 28. 9:: l.J

Cont;r-ol 36.4!1.o

1Incubation mediu.r:i 'B' was used in this study. 'The medium contained

(in~ final volu.~n of 2.7 ml) 5 umoles ADP, 12.5 uncles succinic acid,

i.3 U!llOles Hgso4 , 625 U.'Uoles sucrose, 0.375 ml O.l Holar phosphate pH

7.3 buffer, and 0.4 ml of whole snail 50;.b homogenate. Protein concen­

tration ranged from 32.8-29.2 mg protein/nl homogenate.

2Australorbis glabratus

)Structural formulas of nitroolefins are presented in Table 7

4 Values represent means± standard error for groups of a.t least four

determi_riations.

32

33

TABLb: 7 STRUC'.l.1URAL FOH!:lULAS OF '11HE NITROOLEFINS USED

URI# FOHl'lULA URI# FOHMULA

4 OHH 119 OHH C=C-N02 Cl -C=C-N02

5 -oHH CH3 ~ h -C-C-N02 120 C10HH

c1- ~ h -c-c-1rn2

129

130

- a H 97 OH-l/=c-No2 237

OH7\ H H 99 ve=c-No2

J4

inhibit oxygen uptake indicates a positive correlation. A positivo srun-

ple coefficient of' correlation (0.64j) was calculated when the two ranks

were compared. The population coefficient of correlation was determined

to be statistically different from zero at the 0.05 level of sienificnnce.

r

V. DISCUSSION

These data show that J3-nitrostyrcnes inhibit oxygen uptnke Of whol~

snail homogenates at the concentration used in these studies (J00-440

µMolar). Evidence has been presented by Bovell et al., (196l}) which

indicates that certain J-nitrostyrenes are capable of stimulating respi­

ration and inhibit energy dependent processes of rat liver mitochondria

at concentrations up to 60 µ11olar . At higher concentrations the )3-nitro­

styrenes inhibited respiration as well .

The inability of serine to alter the efficiency of .}-nitrostyrene

as an inhibitor of the DTNB-cysteine reaction indicates that the presence

of equal concentrations of similar compounds possessing -OH and -NH2

groups does not reduce the vulnerability of sulfhydryl compounds to inter­

action with )3-nitrostyrene. The absence of ultraviolet spectral changes

following the addition of excess serine to solutions of ~-nitrostyrena

provides indirect evidence that )3-nitrostyrene does not interact with

serine to the same extent as with cysteine .

F.quimo~ar concentrations of cysteine have been shown to antagonize

the effects of )3-nitrostyrene on the uptake of oxygen by whole snail homo­

genates. The changes obseriied in the ultraviolet spectrum of ]3-nitro­

styrene after the addition of cysteine but not observed with serine suggest

that the antagonism may be due to reaction of cysteine sulfhydryl groups

~ith ~-nitrostyrene . The evidence suggests that.)3-nitrostyrenes inhibit

the uptake of oxygen by interacting with enzymes which require intact

sulfhydryl groups for normal functions .

The absorption of a 1 molar solution of cysteine following the

addition of IJrNB, determined under the conditions of the sulfhydryl assay

(lJ,400) , agrees with the value (lJ,600) published by Ellman (1959) .

35

The data show that the inclusion of EDTA in the suf:hydryl reaction medium

enhances the stabjJ_ity of cysteine in solution at room temperature and

stabilizes the color formed following the reaction of cysteine and m'NB.

EDTA has not been found to produce any qualitative char.~es in the visible

spectrum of the color developed. Ellman (1967) has reported a similar

effect of EDTA on the stability of sulfhydryl compounds in solution.

Since an excess of cysteine abolishes the effect of )3-nitrostyrene

on the color development and excess m'NB is without effect, it is appa­

rent that the concentration of cysteine is t.~e limiting factor in the

progres5 of the reaction. The development of color is progressively de­

layed with increasing nitrostyrene concentration. The results. suggest

that the delay in the developnent of color following the addition of DTNB

to cysteine solutions in the presence of )3-nitrostyrene is due to the

interaction of )3-nitrostyrene with cysteine. Similar effects on the DTNB­

cysteine reaction have been described by Gabay et al. 1 (l968) who has

shmm that the color developlllent of solutions containi:x:ig non-protein sulf­

hydryl material (following addition of DTNB) is delayed in the presence

of protein sufhydryl groups.

A positive correlation (r~.61}5) between the degree of inhibition of

snail homogenate oxygen uptake, by the nitroolefins compared and the

effect of these derivatives on the DTNB-cysteine reaction has been found.

Thus the rate assay procedure described for the est:iir.a.tion of sulfhydryl

reactivity appears to be a useful tool for the evaluation of sul...filyclryl

reactive compounds.

Comparison of molluscicidal activity (Bond, 1968) with oxygen inhi­

bition and sulfhyclryl reactivity data indicates less con·elation between

molluscicidal activity and the in vitro assay data. This may be due to

difforences in the rate of penetration of membrane barriers, biotrans-

formation, distribution end excretion of the ft-nitrostyrenes which may be

independent of the sulfhydryl reactivity of these compounds. However, in

several instances some relation between the j_n vitro evaluation and the

molluscicidal activity of the compounds is discernible. For example:

the · concentration of .8-nitrostyrene found to be lethal to 50-~ of the

exposed snails (LC50) was detormined to be 1.4 parts per million (PPi•l),

while the Lc50 for 4-propionoxy ft-nitrostyrene (URI-llO) was determined

to be 15 PPH. (Bond, 1968) Phenylmercuric chloride was lethal to JO'f, of

the exposed snails at concentrations of O.JPPH. (Bond and Nolan, 19.54)

The saturated derivative of .8-nitrostyrene, 1-phenyl 2-nitroethane (URI-

237) was found to produce 40'% lethality in s11..ails tested at JOOPPL·1 (Bond,

1968). The agreement between the molluscicide data cited and the sulf-

. hydryl assay values for these agents presented in Table 1 is evident.

J7

VI. SUNHARY AND CONCLUSIOr~S

1) J.n interaction between cysteine and ,B-nitrostyrene was observed as ·,

evidenced by alterations in the ultraviolet spectra of J)-nitrostyrene

solutions following the addition of cysteine.

2) A method was devised to permit the use of the color-forming reaction

between DTNB and cysteine as a rate assay for the determination of

sulfhydryl reactivity.

3) The ft-nitrostyrenes studied, were observed to have degrees of sulf­

hydryl activity when assayed under the conditions of this assay.

4) J3-·Nitrostyrene derivatives studied inhibit the oxygen uptake of

homogenates prepared from the snail Australorbis glabratus at the

concentrations used in these studies.

5) The inhibition of oxygen uptake resultin~ from incubation of the

homogenates in the presence of ~ni trostyrene was mitigated in the

presence of cysteine.

6) A positive correlation was found to exist between the effects of

certain_P-nitrostyrenes on oxygen uptake of snail homogenates and

the effect of these compounds on sulf~yd.ryl reactivity as determined

by the ability of the ~-nitrostyrene derivatives to inhibit the reac­

tion between DTNB and cysteine.

JB

VII. REFERE.11CES

Bocobo, F., Curtis, A.C., Block, W.D. and Harel, E.R.: In vitro study of antifungal activity of nitrostyrenas. Proc. Soc. Exp. Biol. Hed. §.g_: 220-222, 1954.

Bond, H. :-;.,:Private Cormnunication, 1968

Bond, H.H., and Nolan, M.O.: Results of laboratory screening tests of chemical compounds for molluscicidal activity II compounds of mercury. Amer. J. Trop. Hed. Hyg. 1= 187-190, 19.54.

Bousquet, E.W.: A pest control composition containing as an essential · active ingredient a compound containing the nitroethylene structure. U.S. Patent 2,335,384, (1$43)

Bovell, c., Packer, L., and Schonbaum, G.: Uncoupling of mitrochondria.l and bacterial respiration by nitrostyrenes and benzal malonitriles. Arch. Biochem. Biophys. 101.i.: 458-467, 1964.

Boyce, c., ~JSsul Jones, T. and van Tongeren, ·w.A.: The molluscicidal activity of N-Tritylmorpholine. 1-lorld Health Organ. Bull. ')1_: 1-11, 1967.

Cason, L.F. and Wanser, sulfides and sulfones.

C.: The preparation of some aryl aminoalkyl J. Amor. Chem. Soc. 2.1= 142-145, 1951.

Cnvins, J. and Friedman, M.: Specific modification of protein sulf­hydryl groups with-'-~unsaturated compounds. J. Biol. Chem. 243: 3357-3360, 1968.

Cort, 1·1.W.: Schistosome dermatitis in the United States (1-ti.chigan). J. Amer. Hed. Ass. 2.Q.: 1027-1029, 1928.

de Villiers, J.P.: Structure and activity in molluscicides: the phenacyl halides, a. group of potentially useful molluscicices. World Health Organ. Bull. g2: 424-427, 1963.

de Villiers, J.P. and Rossouw, 11.: Structure and activity in mollusci­cides. Nature (London) 213: 1208-1209, 1967.

D:b:on, H. : Reactions of lachryma tors with enzymes and proteins. Biochem. Soc. Syrnp. no. 2: 39-49, 1548.

Dixon, H. and Needham, H.: Biological research on chemical warfare agents. Nature (London) 158: 432-438, 1~6.

Ellman, G.: Tissue sufhydryl groups. Arch. Biochem. Biophys. 82: 70-77, 1959.

Ellman, G. and Lysko, H.: Disulfide and sulfhydryl compounds in TCA ex­tracts of hmnan blood and plasma. J. Lab. Clin. Eed. 7.Q: 518-527, 1967.

39

Evans, E.E., Hainos, R.F., Curtis, A.C., Bocobo, F.-D., Block, H.D. and Harrell, E.R.: Evaluation of nitrostyrenes as antifungal agents II. in vivo experiments. . J. Invest. Dermatol. '?]_: · 4)-48, 1956.

Fluharty, A.L. and Saradi, D.R.: On the mechanism of oxidative phos­phorylation VI. localization of the dithiol in oxidative phosphorylation wit..li respect to the oligomycin inhibitory site. Biochemistry£_: 519-522, 196J.

Fryer, H.C.: Concepts and Methods of Experimental Statistics. Allyn and B'.tcon, Inc., Boston, 1966.

Frick, L.P. and Jiminez, W.Q.: Holluscicidal qualities of organo-tin compounds revealed by 6-hour and 24-hour exposures against representative stages and sizes of Australorbis glabratus. World Health Organ. Bull. Jl: 429-431, 1964.

Gabay, s., Cabral, A.M. and Healy, mation of brain sulfhydryl groups. 1081-1085, 1968.

R.: A chemical method for the esti­Proc. Soc. E."{p. Biol. Med. 127:

lam, K.: Sulfhydryl group involvement in a soluble energy transfer factor of the oxidative phosphorylation system. Arch. Biochem. Biophys. 123: e-~2-64 3' 1968.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement wit.~ the folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.

Hackworth, J .F. : Biochem. J. 42

The inhibition of thiol enzymes by lachrymators. 82-90, 1$48.

Nabih, I. and Elvrasimi, H.T.: Structure ·:rnd activity relationships in molluscicides. J. Phann. Sci. 21_: 1202-1206, 1968.

Penefsky, H.S.: phosphorylation.

Partial resolution of the enzymes cata.lyzing oxidative J. Biol. Chem. 2l~2 : .5789-5795, 1967.

Spiegel, H.R.: Theory and Problems of Sta.tistics. Schaum Publishing Co., New York, 1961.

Seiffer, E.A. and Schoff, H.F.: against Australorbis glabratus.

Tests of 15 experimenta.l molluscicides Public Health Rep. 82: 8JJ-8J9, 1967.

Twait, R. c., Muir, R.D. !'.izuba, S. and Crowley, Jr. W.R.: Antifun~al activity of a series of substituted( (nitro alkyl) benzylthio) alkyl amines. J. Hed. Chem. 8: 374-377, 1965 •

. von Brand, T., }';ehl.man, B. and r!olan, H.O.: Influence of some potential molluscicidos on the oxygen consumption of Australorbis glabratus. J. Parasitol. 22.= 475-481, l)J.i-9. ·

l~O

Heinbach, 3.C.: Studios on the intermediary r11etabolism of the aquatic snail Australorbis glabratus. Arch. Biochern. Biophys . h2: 2Jl-244, 1953.

West, E.S., Todd, W.R., Hason, H.S. and van Druggen, J.T.: Textbook of Biochemistry 4th ed. l·~cmillan Co. Neu York, 1966.

Yuki, H., Sano, F., Tnlrn.ma, s. and Suzuki, s.: Studies on antiviral agents I. relationship between chemical reactivity of sulfhydryl reagents and their inactivatine activity of Adenovirus type ). Chom. Pha.rm. Bull. (Japan) 14; 139-146, 1966.

41

VITA

VITA

Robert T. Louis-Ferdin.2nd ·Has born in New York City on August J , •

1937, where he received his preliminary high school and college education.

Following his graduation from college he served in the special service

corps of the U.S. Army as a medical laboratory specialist with the

Medical Research Laboratories , Ft. Yi..nox, Kentucky and the Research

Institute of Environmental Medicine, lfatick , l'~ssachusetts. Upon his

separation from active duty, he accepted a position as a research bio­

cha!.'list at the Research Institute of Environmental Medicine until Septem­

ber , 1966, when he began full t:bne graduate studies at the University

of Rhode Island. He is married to the f ormcr Camille Pugliese of New

York City. They have four children. He is a member of Fhi Sigma, and

Rho Chi.