An Investigation of the Relationship Between the Sulfhydryl Activity and the Toxicity of a Series of Vinyl Sulfone Molluscicidal Agents

A series of vinyl sulfone molluscicidal agents was tested to determine the ability to interfere with: a) the color-forming reaction between cysteine and 5,5 1 -dithiobis {2-nitrobenzoic acid) {DTNB); b) the activity of malic dehydrogenase, a typical sulfllydryl-dependent enzyme; c) the in vitro endogenous respiration in the snail, Australorbis glabratus; and d) the in vivo and in vitro endogenous respiration and pyruvate metabolism of liver, brain, and kidney of albino rats. An approximate intraperitoneal LD~o was :J determined for vinyl sulfone in male and female albino rats. The effect of vinyl sulfone on cysteine treated albino rats · was also observed. Vinyl sulfone and derivatives were found to inhibit the color-forming reaction between cysteine and DTNB indicating sulfhydryl reactivity. A positive correlation between this in vitro sulfhydryl reactivity and molluscicidal potency was observed. However, no significant effect on endogenous respiration was observed in A. glabratus . Similarly, no significant effects on endogenous respiration or pyruvate metabolism were observed in the tissues tested from male and female albino rats. High concentrations of vinyl sulfone '1ere required to significantly inhibit the activity o f malic dehydrogenase.

c) the in vitro endogenous respiration in the snail, Australorbis glabratus; and d) the in vivo and in vitro endogenous respiration and pyruvate metabolism of liver, brain, and kidney of albino rats.
An approximate intraperitoneal LD~o was :J determined for vinyl sulfone in male and female albino rats.
The effect of vinyl sulfone on cysteine treated albino rats · was also observed.
Vinyl sulfone and derivatives were found to inhibit the color-forming reaction between cysteine and DTNB indicating sulfhydryl reactivity. A positive correlation between this in vitro sulfhydryl reactivity and molluscicidal potency was observed. However, no significant effect on endogenous respiration was observed in A. glabratus . Similarly, no significant effects on endogenous respiration or pyruvate metabolism were observed in the tissues tested fr om male and female albino rats. High concentrations of vinyl sulfone '1ere required to significantly inhibit the activity o f malic dehydrogenase.
iii An intraperitoneal LD 50 of 3 mg/kg vinyl sulfone was found for male and female albino rats. A sublethal dose of vinyl sulfone was found to cause increased blood urea nitrogen and decreased urinary output in albino rats while producing no significant effects on SGOT, SGPT, and hematocri t levels.
These data indicate that the toxicity of vinyl sulfone and derivatives is related to sulfhydryl reactivity; however, this toxicity was not due to decreased endogenous respiration in either A. glabratus or albino rats. The toxicity of sublethal doses of vinyl sulfone was postulated to be caused by the impairment of normal renal function.
A hypothesis wa s proposed that the toxicity of vinyl sulfone and deriva~iv es i n A. glabratus may be due to the reaction of these agents with integral membrane proteins responsible for normal cellular function.
i I would also like to thank my parents, Mr. and Mrs.
Rudolph La Rocca, for their guidance and financial help.
I would also like to express my gratitude to Dr.
H. Katzin whose surgical skill has given me new sight.

8.
Endogenous respiration in brain slices of female rats treated in vivo with vinyl sul- Specific chemotherapy for the treatment of schistosomiasis is effective in the early stages of the disease.
However, it is of little value and may even be dangerous in the advanced stages. This presents a major problem because most geographical areas affected by the disease are characterized by an agricultural population poor in resources, and thus early medical detection and care is impossible.
One approach in the attempt to control schistosomiasis has been eradication of the intermediary sna il hosts through the use of molluscicides. The use of molluscicid a l a g -7 ents presents a number of uniquely difficult problems. Molluscicidal a g ents must be toxic in low concentrations to the molluscan host and, at the s ame time , non-toxic to ma n and . ' domestic and wild animals. The agents should also be sufficiently soluble in water, inexpensive and easy to use.
According to the World Health Organization (1965), progress in the control of schistosomiasis has been slow due to the failure to recognize · its public heal th importance earlier, a dearth of basic data on the prevalence and transmission of the disease, the inadequacy of available control measures and slow progress in developing and evaluating new and better ones, the small number of trained personnel available, and the lack of money.
In recent years the importance of molluscicides as a means of controlling the snail vectors of schistosomiasis has been well established. Over the years the pesticide industry has developed numerous chemicals, certain of which possess molluscicidal properties. The basis, however, on which compounds are selected for testing as molluscicides remains largely empirical and any approach that enables selection to be made with a better understanding of molluscicidal activity would be an advance.
The bulk of the data available concerning molluscicidal agents which are presently in use or in the last screening stages deals with mortality times and concentrations required for these results. Little information concerning the effects of the drug on man or animals is available and far less information is available concerning the mechanism of action of these compounds.
· N-Tritylmorpholine has been shown to possess outstanding molluscicidal activity (Boyce ~al. 1966). deVilliers (1963) has shown that phenacyl halides are also a potenti-ally excellent group for use as molluscicidal agents.
The need for an adequate molluscicide is so great, and the search so extensive, that man has gambled with the ecological balance of some geographical areas by introducing foreign organisms which attack and kill the intermediary snail host of schistosomiasis. Berg (1971) (Conn and Stumpf, 1967). Sulfhydryl groups have been shown to be required for membrane integrity and function. Knauf and Rothstein (1971) have shown that the organic mercurial para-chloromercuriphenylsulfonic acid (PCMBS) inhibits the active transport of ions in red blood cell membranes and suggested that the mechanism of action is related to the effect of the mercurial on the sulfhydry l groups of the N~-K activated ATPase system.
Hegener (1968) has presented evidence that a number of mercurials cause an increase in lysosomal membrane permeability by reacting wi t h membrane sulfhydryl groups. Watkins e t __ al.
(1970) have shown that alloxan and other sulfhydryl reagents cause an increa s e in the permeability of toadfish be t a islet cells to mannitol and have suggested that they may damage cell membranes by combining with sulfhydryl groups essential for their integrity. Solberg and Forte (1971) have shown that sulfhydryl reagents decrease hydrogen ion secretion in the ·isolated frog gastric mucosa. Mackworth (1946) has shown that the lachrymatory effect of certain war gases is due to the reaction of these compounds ~ith thiol enzymes such as succinic "ctehydro genase, choline dehydrogenase, yeast alcohol dehydrogenase, papain, pyruvic oxidase, triosephosphate dehydrogenase, xanthine oxidase, and carboxylase. Chang et al. (1970) 16 have confirmed the evidence that various sulfhydryl reagents cause conduction blockade of the motor axon, depolarization of the muscle membrane, sustained contraction, and depression and prolongation of the muscle 8ction potential in the rat phrenic nerve-diaphragm preparation. These results point toward an active role for sul:fhydryl groups in neuronal pathways. Landon and Forte (1971) have reviewed evidence indicating that a membrane bound N~-K ATPase system is associated with the transport process of N~ and K in mammalian cells.
It has been postulated that this system participates in renal tubular reabsorption of N~. Lehninger (1971) has cited evidence demonstrating that the ATPase activity of myosin is dependent on the integrity of specific sulfhydryl groups. Organic mercurial diuretics have been shown to inhibit sodium-potassium dependent ATPase activity whereas non-diuretic mercurials have been shown to have no effect on Na+-K+ dependent ATPase activity (L~ndon and -Forte, 1971). Because the kidney accumulates the highest tissue levels of mercury (mercury is a sulfhydryl reagent with a high equilibrium affinity constant for the reaction between the S atom of a thio group and mercury) sulfhydryl groups are believed to play an important role in normal renal function (Clarkson, 1972 Evidence has accumulated indicating that pulmonates excrete uric acid via an organ which functions in a manner similar to the human kidney (Picken, 1937;Muller, 1963). Potts (1967Potts ( ,1968  They investigated the toxicity of vinyl sulfone on the basis of the theory that mustard gas may be oxidized to mustard sulfone or vinyl sulfone. Toxicity was studied in mice and rats which had been administered the compounds ~ither cutaneously or in- travenously.
An intravenous LD 50 of 11 mg/kg for mice and 12 mg/kg for rats was reported. High doses were reported to produce pulmonary edema and death in rats within twentyfour hours.
Marked parasympathomimetic activity terminating in clonic convulsions and death were reported on intravenous administration of vinyl sulfone to rabbits. Cordier and Cordier (1950) st udied the effects of oxidati on products of mustard gas on the cardiovascular system in order to dete rmine whether the atropine like effects and toxicity of mustard gas were due to the intact molecule or a breakdmm product of mustard gas. Vinyl s ulfone produced transient hypertension followed by prolonged hypotension (sometimes terminating in death) and had no effect on salivary secretions. It was therefore determined that the effects of mustard gas were due to the intact molecule.
Ford-Moore et al . (1949) have cited evidence which the y b e lieve indicates that the reaction between sulfhydryl gro , and compounds such as vinyl sulfone may be reversible und r biological conditions. The basis of this belief is that the thioether linkage o~ the reaction between vinyl sulfone and cysteine can be split, at slightly alkaline pH, with silver salts .
The sulfhydryl reactivity of vinyl sulfone is well documented (Ford-Moore et al. 1950;Buckley et al. 1947;. Ford-Moore et al. 1949;and Yuki et al . 1966), and its pharmacological and toxicological properties have been linked with this reactivity. Grant et al. (1946) studied the effect of mustard gas and vinyl sulfone on the activity of urease, a sulfhydryl dependent enzyme . Vinyl sulfone was found to inactj_va te the enzyme between pH 6 and pH 8 ~ and its effect was linked to dissociation characteristics and reactivity of the sulfhydryl groups with vinyl sulfone at those pH's. Watkins ~ al. ( 1952) fouJ1d that hemolytic complement is rapidly inactivated by small amounts of vinyl sulfone or S-mustard . Thiosulfite was found to protect against both substances.
Inactivation was thought to be the result of reaction between vinyl sulfone and the sulfhydryl groups of complement. Peters and Wakelin (1949) studied the effects of mustard gas, dichlorodiethyl sulfone, and vinyl sulfone on the pyruvic oxidase system in pigeon brain tissue. The system was studied because it was a known sulfhydryl dependent enzyme system, and their initial hypothesis was that vesication was due to damage of sulfhydryl dependent enzymes.
Dichlorodiethyl sulfone and vinyl sulfone were found to inhibit the pyruvic o xidase system at concentrations of l0-4 H. On a molar basis, vinyl sulfone was found to be the more toxic.
Vinyl sulfone had no effect on cyto- In all cases the toxicity of vinyl sulfone has been linked to its sulfhydryl reactivity.
Vinyl sulfone and its derivatives exhibit molluscicidal activity (Bond, 1971) and possess an electron withdrawing so 2 group with an alpha-beta unsaturation directly adjacent to it. The structure then of vinyl sulfone is similar to that of the beta-nitrostyrenes. The fact that both classes of compounds are molluscicidal and both contain structural portions which are very similar leads to the hypothesis that vinyl sulfone ma y exert biological activity via a mechanism similar to t h at of the beta-nitrostyrenes.
This study has been designed to determine whether the vinyl sulfones exert any effect on respiration in A.
glabratus or in rats and whether their toxicities can be correlated with sulfhy dryl reactivity and decre a sed respirati on.  (Ochoa, 1955). Immediately after the equilibration, oxygen uptake was monitored every ten minutes for one hour. Following the measurement of oxygen uptake, the whole snail was removed, placed into a pre-weighed aluminum dish, and dried at 90 to 100°C for twenty-four hours. The dry weight of the t issue was calculated and the endogenous respiration expressed as the Q0 2 (microliters oxygen/mg dry tissue/hour).  ing to the method of Reitman and Frankel (1957).

DETERMINATION OF AN
Female rats, weighing 125-150 grams, were treated intraperitoneally with either vinyl sulfone (2 mg/kg) or 5% ethanol -(1 ml/kg) and placed in individual metabolism cages with free access to food and water. Three days after treatment the animals were bled, via tail slash, and the blood collected.
One ml of the appropriate substrate was then placed into a test tube incubated at J7°C. Two-tenths ml of serum were added and the tube allowed to incubate for thirty minutes. Samples used for the determination of SGOT were incubated for sixty minutes.
Following the incubation period, one ml of 2,4dinitrophenylhydrazine was added to each tube. The samples were allowed to stand at room temperature for twenty minutes and then ten ml of o.4N sodium hydroxide were added to each tube. Reagent and tissue blanks were run for each analysis in the same manner. After thirty minutes the a~ sorbance of each sample was determined spectrophotometrically at 520 nanometers against a water blank.
A standard curve was run simultaneously with the treated and control samples and SGOT and SGPT levels were calcul ated from the graph of the standard curve.

b. DETERMINATION OF BLOOD UREA NITROGEN
Blood urea nitrogen was determined using the diacetyl monoxime method of Rosenthal (1955 The degrees of freedom were taken as n 1 + n 2 -2 . The level of significance (p) was determined by compari s on of "t" with values from standard tables.  (Dixon and Massey, 1951).

A. SULFHYDRYL REACTIVITY
The reaction between 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and a compound containing a sul:fhydryl group is presented in Figure 1. The anion yielded in the reaction is yellow and the intensity of the yellow color can be used to determine the extent of reaction between DTNB and the sul:fhydryl compound. Table 1   The data in Table 2 represents the relationship be-     .+:-   Expr e ssed as micromoles cysteine react ed/ mi cromoles inhibitor . The effect of substrate (oxalacetate) concentration on the in vitro activity of MDH is shown in Table 5. A dose dependent r elationship exists between malic dehydrogenase activi ty and oxalacetate concentration. Increasing the concentration of oxalacetate from 1.9 x 10-JM to   Table 6. The effect of in vitro vinyl sulfone on the endogenous respiration of liver and brain slices of female rats is presented in Table 7. A concentration of J.J x 10-JM vinyl sulfone produces no effect on endogenous respiration while a concentration of 1.7 x 10-JM vinyl sulfone exhibits significant (P 0.05) inhibitory effects on endogenous respiration in brain slices while producing no effect on endogenous respiration in liver slices . Table 8 shows endogenous respiration in brain slices of female rats treated in vivo with vinyl sulfone.
An intraperitoneal dose of 20 mg/kg vinyl sulfone produces no significant change in endogenous respiration JO minutes after treatment.
The effect of in vitro vinyl sulfone on pyruvate metabolism in brain homogenates of male rats is shown in Table 9. A concentration of 9.7 -4 x 10 M vinyl sulfone produces a significant (P 0.05) 53% decrease in pyruvate metabolism. Table 10 represents the effect of in vitro  Expressed as the Q02· b.
Dissolved in absolute ethanol . c.
Represents the mean and standard deviation of four determinations run in duplicate. d.
Represents the mean and standard error of five determinations run in duplicate. e.
Absolute ethanol used as control.
An intraperitoneal dose of 10 mg/kg produces a significant (P 0.05) increase in the hematocrit of male rats 90 minutes after treatment. Concurrent with the increase in hematocrit in male rats, a copius edema of the peritoneal cavity is observed.
The hepatotoxicity of vinyl sulfone in female rats is shown in Table 15. An intraper itoneal dose of 2 mg/kg vinyl sulfone does not produce any significant change in the serum glutamic pyruvate transaminase or serum glutamic oxalacetic transaminase levels 72 hours after treatment.  Adjusted to pH 7 .3 with NaOH and administered fortyfive minutes prior to the intraperitoneal administration of vinyl sulfone . c. Adjusted to pH 7.J with NaOH and administered fiften minutes prior to the intraperitoneal administration of vinyl sulfone.   V.

DISCUSSION
These data indicate that the mechanism of toxicity of vinyl sulfone in A. g1abratus and albino rats is not . the inhibition of endogenous respiration in the tissues tes~ed and that the mechanism of toxicity of vinyl sulfone is probably related to its reactivity with other important sulfhydryl groups.
A structural comparison of the three aliphatic com- The halogenated vinyl sulfone, URI #7 9J, possesses significantly greater sulfhydryl reactivity and molluscicidal pot ency than URI #744. It seems likely that the halogenation of the alpha-beta carbon atoms of various derivatives of vinyl sulfone would also increase the sulf-hydryl reactivity and molluscicidal potency in the same manner as the halogenation of the phenyl substituted compound.
In general, it appears that, in this series of compounds, sul:fhydryl reactivity and molluscicidal potency are Malic dehydrogenase (MDH) is classified as a type A sul:fhydryl enzyme by Webb (1966) and Pfleiderer (1969). Fondy et al . (1970) have shown that sul:fhydryl groups exist at or near the active center of MDH and Gregory ~ al.
(1971) have shown that MDH has at least two (and possibly three) sulfhydryl groups at or near the enzymatic active centers which are essential for activity. Anderton (1970) has shown that MDH contains at least 12-14 sul:fhydryl groups.
Since MDH is a sul:fhydryl dependent enzyme, it is of interest to determine if vinyl sulfone inhibits the activity of MDH. The inhibitory effect of vinyl sulfone is dose dependent as seen in Table 4 In the light of the above discussion, and the fact that Peters and Wake1in (1949) reported that the inhibitory action of vinyl sulfone on succinic, malic, and lactic dehydrogenase was low in comparison to its effect on pyruvate dehydrogenase, the inhibitory effect of vinyl sulfone on the sulfhydryl dependent enzyme MDH can be considered to be insignificant.
The The stat istical1y significant decrease in in vitro endogenous respiration of brain slices of female rats, and in the in vitro pyruvate metabolism of liver and brain homogenates of male rats, when treated with 10-JM vinyl sulfone, is of questionable pharmacological significance.
The concentration required for activity would probably never be reached in the in vivo intracellular situation.
The above data and the fact that no effect was seen on the endogenous re spi ration of liver slices of female rats or in the endogenous respiration and pyruvate metabolism of animals treated in vivo with vinyl sulfone, suggest that the mechanism of toxicity of vinyl sulfone in albino rats is not the inhibition of cellular respiration.
Vinyl sulfone is extremely toxic to albino rats. Anslow et al. (1947)  The impairment of renal function by sublethal doses of vinyl sulfone is not surprising since sulfhydryl containing enzymes are involved in many of the tubul a r transport mechanisms (Guyton, 1971). Clarkson (1972) cites evidence indicating that the kidney accumulates high concentrations of mercury which is a sulfhydryl reagent with a high affinity for the thiol groups involved in renal function. Landon and Forte (1971) have supported these observ ations by reviewing evidence for a membrane bound 70 Na+-K+ activated ATPase system associated with renal trans-+ + port of Na and K • ATPase systems have been shown to be dependent on sul:fhydryl groups.
As stated previously, these data lead to the view that vinyl sulfone does not exert its toxic effect as a result of the inhibition of endogenous respiration via the blockage of sul:fhydryl dependent enzymes involved with the process but that sul:fhydryl reactivity and toxicity are correlated in some other manner.
The convulsions seen by Anslow et al. (1947), and in our investigations, following high doses of vinyl sulfone are probably due to CNS toxicity. The rapid death of albino rats, preceded by an _ increase in hemat ocrit, is probably due to changes in blood volume caused by the vesicant activity of vinyl sulfone while the increased blood urea nitrogen and decreased urinary output {volume) produced by sublethal doses of vinyl sulfone is probably due to the reaction of vinyl sulfone with integral membrane proteins involved in renal function.
It is difficult to believe that vinyl sulfone could act at the intracellular level when used as a rnolluscicide which is diluted, in large expanses of water, to a concentration of l0-7 M. It is not difficult to hypothesize, however, that the toxic action of vinyl sulfone on A.
glabratus may be due to its reactivity with sulfhydryl groups of integral membrane proteins involved with normal cellular function. Some data which supports this hypothesis are: a) vinyl sulfone does exhibit a high degree of vesicant activity and the toxicity of vinyl sulfone has been correlated with its sulfhydryl reactivity; b) Louis-Ferdinand (1968) has reported that some beta-nitrostyrenes exhibit potent molluscicidal activity while producing little or no inhibition of oxygen uptake in snail homogenates; c) the concentration of vinyl sulfone required to inhibit a typical sulfhydryl dependent enzyme involved in cellular respiration is so high that it is unlikely that such a concentration could be reached in the in vivo situation unless the compound were concentrated by a particular physiological process; d) vinyl sulfone has been shown to exhibit a high degree of reactivity with hydroxyl and amino groups of proteinaceous compounds (Ford-Moore et al.

1950).
de Villiers (1963) has stated that most molluscicides exhibit a mode of action characterized by either interference with the osmoregulatory system of the snail or interference with enzyme systems which are dependent on sulfhydryl groups . In view of this statement, and the above hypothesis, it seems possible that the toxic effect of vinyl sulfone on A. glabrattls is produced by its reactivity with membrane bound proteins. This toxicity could be manifested in many ways. It is possible that, similar to the action of sublethal doses in albino rats, vinyl sulfone causes impairment of renal function in the snail and prevents the normal excretion of uric acid. It is also possible that vinyl sulfone produces changes in electrolyte balances due to its vesicant activity.
VI. SUMMARY AND CONCLUSIONS 1. Vinyl sulfone and derivatives exhibit a high degree of sulfhydryl reactivity which is correlated with molluscicidal potency.
2. High concentrations of vinyl sulfone are necessary to inhibit malic dehydrogenase.
J. ~he toxic action of vinyl sulfone on A. glabratus and albino rats is not due to the inhibition of sulfhydryl dependent enzymes involved in cellular respiration.
The concentration required to achieve such an effect is so high that it is unlikely that this concentration would be reached in the in vivo intracellular situation.
4. Vinyl · sulfone is extremely toxic to albino rats and this toxicity is related to its sulfhydryl reactivity.
5. The toxic effect of sublethal doses of vinyl sulfone in albino rats is caused by impairment of renal function leading to uremia while the toxic effect of doses of vinyl sulfone J.J times greater than the LD 50 do~e is probably due to hypovolemia caused by the vesicant · activity of vinyl sulfone in the peritoneal cavity.
6. An hypothesis is made that the molluscicidal activity of vinyl sulfone and derivatives may be due to the reactivity of these compounds with integral membrane proteins involved in normal cell function. VII.