CHAPTER IX.

_PHYSIOLOGY OF POISONING_ (_continued_).

PROTEOLYTIC, CYTOLYTIC, BACTERIOLYTIC AND VARIOUS DIASTASIC ACTIONS OF VENOMS: DIASTASIC AND CELLULAR ACTION ON VENOMS.

A.--PROTEOLYTIC ACTION.

The proteolytic action of venoms on gelatine, fibrin, and egg-albumen has been studied by Flexner and Noguchi,[53] Delezenne,[54] and subsequently by Noc[55] in my laboratory. It was already known that _in vivo_ certain venoms exert a manifestly dissolving action on the endothelium of blood-vessels and on the muscular tissues themselves.

Delezenne, on his part, has established the existence in snake-venoms of a _kinase_ analogous to the kinase of leucocytes and enterokinase. Venom alone does not attack egg-albumen coagulated by heat, but it confers an exceedingly strong digestive power on inert pancreatic juices.

_Lachesis_-venom has been found to be much the richest in kinase. It digests gelatine perfectly, and when this substance has been subjected to its action it is no longer capable of being solidified.

_Lannoy_,[56] on the other hand, experimenting upon albuminoid substances (casein, albumins of ox-serum) in solution, has shown that _Cobra_-venom and that of _Vipera_ disintegrate the albuminoid molecule; but the latter remains soluble after the addition of formol and is no longer precipitable by acetic acid. The hydrolysis never leads to the stage of peptone, but only to the formation of albumoses which give biuret-reaction.

The action of venoms upon fibrin may be demonstrated _in vitro_ by bringing sufficient quantities of venom, 1 centigramme, for example, into contact with small fragments of non-heated fibrin, derived from blood clots from an ox, rabbit, or birds, and carefully washed. These fragments soon separate from each other, and become dissolved in a space of time which varies according to the venom used. The VIPERINE-venoms, especially those of _Lachesis_ and _Ancistrodon_, are the most active. _Viper_-venom is much less so, and the venoms of COLUBRIDÆ are the slowest.

This proteolytic action of the various venoms corresponds pretty exactly to their coagulant and decoagulant action on rabbit- or horse-plasma, so that, as I have already stated, we must suppose that the property possessed by VIPERINE-venoms of more or less rapidly dissolving blood which they have caused to coagulate, results from the fact that these venoms contain, in addition to a coagulant substance, another substance which is strongly proteolytic.

The latter is destroyed by heating. _Lachesis_-venom, when heated to 70° C., no longer has any dissolving action on either gelatine or fibrin. Moreover, antivenomous serum furnished by horses vaccinated against heated venoms does not prevent proteolysis by non-heated venoms. On the other hand, the serum of animals vaccinated against VIPERINE-venoms, simply filtered by the Chamberland process and non-heated, affords perfect protection to gelatine and fibrin against the dissolving action of these venoms.

B.--CYTOLYTIC ACTION.

Simon Flexner and Noguchi[57] have observed that the venoms of _Naja_, _Ancistrodon_, _Crotalus_, _Vipera russellii_, and _Lachesis flavoviridis_, contain substances which possess the property of dissolving a large number of the cells of warm-blooded and cold-blooded animals, and that these _cytolysins_ are very markedly resistant to high temperatures.

They employed for their experiments 5 per cent. emulsions of organs, spermatozoids, or ova in physiological saline solution. The solution of venom at a strength of 1 per cent. was kept in contact with the different kinds of cells for three hours at a temperature of 0° C.; the liquid was then centrifuged and examined with the naked eye and under the microscope.

The venoms experimented upon dissolved more or less rapidly the parenchymatous cells of the liver, kidney and testicle of the dog, guinea-pig, rabbit, rat and sheep. The most active venoms in this respect were those of _Vipera russellii_, _Ancistrodon_ and the _Cobra_; the venom of _Crotalus_ was the least active.

With regard to the nerve-cells, spermatozoids and ova of cold-blooded animals (frogs, fish, arthropods, worms, and echinoderms) _Cobra_-venom proved to be the most active; then that of _Ancistrodon_, and lastly that of _Crotalus_.

These cytolysins are not destroyed by heating for thirty minutes at 85° C. in a damp medium, nor by dry heating for fifty minutes at 100° C.

C.--BACTERIOLYTIC ACTION.

If we bring into contact with a 1 per cent. solution of _Cobra_-venom, rendered aseptic by filtration through porcelain, sensitive micro-organisms, such as the cholera vibrio, or the bacterium of anthrax in a very young non-sporulated culture, or in its non-spore-producing variety, we find that these microbes are dissolved by the solution of venom in varying periods of time.

On making a direct microscopical examination we see that Koch’s vibrios become immovable, then break up into granulations and disappear in the liquid. The bacteriolysis is even more distinct in the case of the bacterium. The enveloping membrane seems to dissolve, and the microbe appears as though composed of a series of granulations placed end to end, which finally disperse and disappear.

By my instructions this bacteriolytic property of venom with respect to different species of micro-organisms was studied by Noc. It was especially clearly seen with the non-spore-producing bacterium of anthrax, the cholera vibrio, _Staphylococcus aureus_, the bacillus of diphtheria, and _B. subtilis_ in a young culture; it is less distinct with _B. pestis_, _B. coli_, and _B. typhi_, is almost _nil_ with the pyocyanic bacillus and _B. prodigiosus_, and _nil_ with _B. tuberculosis_.

Investigations have likewise been made by Noc, and subsequently by Goebel,[58] in order to determine whether cobra-venom dissolves Trypanosomes. These hæmatozoa are more resistant than bacteria, but they nevertheless end by being dissolved after twenty to thirty minutes’ contact in the 1 per cent. solution.

The bacteriolytic substance in venom is distinct from that which produces proteolysis, for the latter is destroyed at 80° C., while the former only disappears with a temperature of and beyond 85° C. maintained for half an hour. It is likewise distinct from the hæmolysin, for this resists temperatures considerably higher than 85° C. Moreover, venom which has dissolved microbes until the saturation point has been reached, is found to have preserved in its entirety its hæmolytic power upon the red corpuscles of the horse. Neither does it act upon the microbes owing to the presence of a _cytase_ or _alexin_. The well-known characteristics of alexins are not met with here--destruction at 55° to 56° C., sensitivity to light, rapid alteration at ordinary temperatures, &c.

We cannot, again, compare the bacteriolytic action of venom to that of rat-serum, which dissolves _B. anthracis_ by aid of a substance distinct from vibrionicide alexin. According to the researches of Malvoz and Y. Pirenne, the lysin of rat-serum appears to be a basic substance, the neutralisation of which destroys its activity. Now _Cobra_-venom in a very active solution is perfectly neutral to sensitive litmus papers, while these are turned blue by rat-serum. Moreover, venom acts not only upon microbes of the same kind, but also on very different species which are not affected by rat-serum, especially upon _B. pestis_, for which, on the contrary, this serum, when fresh, proves a favourable culture medium. The bacteriolytic power of _Cobra_-venom therefore constitutes a special property of venom.

“In their work on the cytolysins of venom, S. Flexner and Noguchi have shown that animal cells, when heated to 55° C. and rendered inactive, do not undergo complete dissolution under the influence of venoms which destroy the fresh cells. The authors in question infer the existence of cellular receptors (_endo-complements_, according to the theory of Ehrlich), which fix the amboceptors of venom. Pursuing the same order of ideas, I have observed that bacteria killed by heating for one hour at 60° C. do not undergo total disintegration as do living bacteria. But, while Flexner and Noguchi infer the plurality of the cytolysins in venom for different animal cells, I have not been able to prove the same thing with regard to the bacteriolysin; venom saturated with cholera vibrios to such an extent that vibrios added at repeated intervals are no longer dissolved, is incapable of dissolving another highly sensitive species of microbe, such as the asporogenous bacterium, and _vice versâ_. Besides, it would be difficult to understand the existence in venom of cytolysins specific for a whole series of species of micro-organisms” (Noc).[59]

Antivenomous serum, in a dose of 0·01 or 0·05 c.c., neutralizes the bacteriolytic action of 1 milligramme of _Cobra_-venom, while normal serum heated, even in larger doses, is without effect. The lysin and the antivenomous serum appear also to enter into stable combination; by heating to 80° C., after dilution of the mixture neutral antivenomous serum + venom, the property of dissolving is not restored to the latter.

Pursuing his researches upon the bacteriolytic actions, Noc has also shown that the _fresh_ serums of the rabbit, horse, guinea-pig, rat, and man are capable of destroying them completely. We must conclude from this that venom has the property of fixing the alexin of these fresh serums, and in fact it is easy to show that this fixation takes place by experimenting with hæmolytic alexin, which is much more easy to study; it is sufficient to eliminate the intervention of the hæmolysin proper to _Cobra_-venom.

With this object, Noc employed horse-corpuscles (which are readily dissolved by fresh rat-serum), and neutralised the hæmolysin proper to the venom by antivenomous serum, which has no effect upon fresh horse-corpuscles and upon the alexin of rat-serum.

For experimental purposes six tubes are prepared with contents as follows:--

(1) 0·5 c.c. of fresh rat-serum.

(2) 0·5 c.c. of fresh rat-serum + 0·5 milligramme of Cobra-venom (0·5 c.c. of a solution of 1 in 1,000).

(3) 0·5 c.c. of fresh rat-serum + 1 milligramme of venom (after fifteen minutes’ contact of the venom with the alexin in tubes 2 and 3 the venom is neutralised by 1 c.c. of antivenomous serum in the case of tube 2, and by 2 c.c. in that of tube 3).

(4) 1 milligramme of venom.

(5) 1 c.c. of antivenomous serum.

(6) 0·5 c.c. of fresh rat-serum + 1 c.c. of antivenomous serum.

To each tube 2 drops of defibrinated horse-blood are added, and the tubes are placed in the stove at a temperature of 35° C.

In tubes 1 and 6, which contain fresh rat-serum alone, and fresh serum + antivenomous serum, hæmolysis appears in a few minutes. In tube 4, which received venom alone, hæmolysis is also produced in one hour. It is not produced at all in tubes 2 and 3, which received the neutral mixture of fresh serum and venom, proving that the hæmolytic alexin has been fixed by the venom. The latter, therefore, here plays the part of a true fixator or _amboceptor_.

Venom behaves, in short, after the manner of extracts of organs. The fixation of hæmolytic alexin by extracts of organs, the tissues, and animal cells (liver, spleen, spermatozoids, &c., &c.), has already been demonstrated by V. Dungern, P. Müller, Levaditi, and E. Hoke. The same fact is also observed with solutions of peptone. The fixation of alexin is therefore a general property of certain albuminoid molecules.

It was interesting to endeavour to reproduce, with _Cobra_-venom, J. Bordet’s experiments upon alexins and anti-alexins. It was to be hoped that we had in this substance an anti-alexic body capable of being preserved for an indefinite time and constant in its activity, which would enable us easily to measure the dose of alexin contained in a small quantity of a serum, or other liquid of leucocytic origin.

The experiment proved to Noc that, contrary to the ideas of Ehrlich and his pupils, and conformably to the results obtained by Bordet with serums and toxins, the neutralisation of venom takes place in a variable ratio.

If a dose A of fresh serum is capable of neutralising exactly 5 milligrammes of _Cobra_-venom with regard to a sensitive microbe, on employing a dose of the strength of 2 A we ought to find a bactericidal dose, 1 A, in the excess of serum, according to the theory of definite proportions. No such bactericidal action is seen, however; the serum, on the other hand, acts in the contrary direction by means of its nutritive substances, and in the mixture 2 A + _venom_ we obtain a larger number of colonies of micro-organisms than in the mixture A + venom.

We see, then, that the property of cells of fixing in excess the active substance in serums, discovered by Bordet for the hæmolysins (staining phenomena), is met with again in the case of extracts of organs, at least with regard to the bacteriolytic substance of _Cobra_-venom.

It results, then, from the foregoing facts that _Cobra-venom contains a cytolysin, which acts upon micro-organisms and is capable of fixing the alexin of normal serums_.

The application of these data to the living animal is evidently full of difficulties, by reason of the complexity of the substances that come into play. Let us see, however, to what extent they are capable of serving to explain the phenomena that are produced as the result of poisoning.

It was observed by Kaufmann that the cadavers of animals which have died from snake-bite are very rapidly invaded by the bacteria of putrefaction. Welch and Ewing, referring to these phenomena of rapid putrefaction in cases of death from venom, explained them as being due to the loss of the bactericidal power of the serum. In hot countries, even when snake-bites are not fatal, they are frequently complicated by local suppuration or gangrene, occasioned by micro-organisms introduced at the time of the bite. The minute analysis of the phenomena of poisoning shows, in reality, that the organism undergoes different modifications according to the quantity of venom injected and its channel of penetration.

When the dose of venom is rapidly lethal, whether because it penetrates into the veins or because a larger amount of it is diffused beneath the skin, it occasions a transient hypoleucocytosis, which is, moreover, a reaction common to injections of venom, pro-peptone, extracts of organs, and microbic toxins (Delezenne, Nolf). It follows that blood collected a short time after the injection may be totally bereft of its bactericidal power, in consequence of the disappearance of the leucocytes, which have migrated into the organs.

Thus it was observed by S. Flexner and H. Noguchi that the serum of a rabbit, treated with 10 milligrammes of _Cobra_-venom, showed, fifty-seven minutes after the injection, a great loss of bactericidal properties. But it is impossible to conclude, from the diminution of bactericidal power in this experiment, that the alexin becomes fixed by the venom. Since the secretion of alexin is connected with the presence of leucocytes, the hypoleucocytosis due to the venom is sufficient to explain the loss of bactericidal power.

Nevertheless, the action of venom is not confined to these physiological phenomena; in diffusing itself through the organism it stays more especially in parts where the circulation has become slower, in the capillaries of the organs where the leucocytes that have disappeared from the general circulation are already to be found agglomerated and altered. Here the cytolysins of the venom, continuing their effects, are capable of neutralising the alexins set at liberty by the destruction of the leucocytes, and thus the rapid multiplication of the bacteria of putrefaction, which have come from the intestine or were carried in with the bite, is easily explained. In the same way, we can account for the suppuration that is met with as a complication of non-lethal bites, in spite of the hyperleucocytosis consequent upon the penetration of a weak dose of venom; immediate neutralisation of the alexin set at liberty at the level of the wound has sufficed to enable micro-organisms to multiply.

D.--VARIOUS DIASTASIC ACTIONS OF VENOMS.

So long ago as 1884, de Lacerda, in his “Leçons sur le venin des serpents du Brésil,” described the results of his researches upon the diastasic actions of venom. He proved that venom emulsifies fats, causes milk to curdle, and does not saccharise starch. But the solutions of venom employed by this author were not sterile, so that putrefactive phenomena may be believed to have occurred in the course of his experiments.

The subject has been studied afresh by Wehrmann[60] in my laboratory, and afterwards by Lannoy.[61] These two investigators have shown that venoms do not hydrolyse either starch or inulin. _Cobra_-venom and that of _Vipera_ change saccharose very slightly. They do not modify the glucosides (amygdalin, coniferin, salicin, arbutin, and digitalin); they therefore do not contain emulsin.

On the other hand, these venoms possess, as I have already stated, very interesting _kinasic_ properties, which have been pointed out by Delezenne.[62] They consist in the fact that while venom alone is incapable of digesting cooked albumin, we have only to add to a pancreatic juice, in itself devoid of effect upon albumin, a trace of venom, to see this albumin immediately become digested.

_Lachesis_-venom is especially active in this respect. In Delezenne’s experiments it was generally sufficient to add to 1 c.c. of inactive pancreatic juice, 0·5 to 1 c.c. of a 1 in 1,000 solution, that is 0·5 to 1 milligramme of venom, to obtain the digestion of a cube of albumin weighing 0·5 gramme in the space of from ten to twelve hours. Much weaker doses, ⅕, ⅒, sometimes even 1/80 of a milligramme still gave the same result, with this sole difference that digestion took twenty-four, forty-eight, and even seventy-two hours to become complete.

_Cobra_-venom was found to be a little less active than the foregoing, but still its action was usually evident enough when it was employed in a dose of 0·5 milligramme or even 0·1 milligramme. As for the venom of _Vipera berus_, it was often necessary to employ it in doses from five to ten times stronger in order to obtain the same result.

Delezenne has ascertained, on the other hand, that these venoms entirely lose their kinasic power when they are subjected to ebullition for fifteen minutes.

This _kinase_ or diastase, capable of quickening the inert pancreatic juice, must evidently be of very great utility to the reptile in enabling it to digest its prey. Venom, therefore, is not, as has long been believed, a purely defensive secretion; it corresponds to a physiological necessity, like that of the intestinal or pancreatic juice. Herein is to be found the explanation of the fact that the non-poisonous snakes, although destitute of organs of inoculation, possess supralabial or parotid glands which produce venomous saliva.

Experiments have been made by Ch. Féré[63] to determine the effect upon the development of the embryo of the introduction of venom into the albumen of the egg of the fowl. He found that 83 per cent. of the embryos, developed in eggs intoxicated with 0·05 milligramme of viper-venom and opened after being incubated for seventy-two hours, exhibited various anomalies in development.

E.--ACTION OF VARIOUS DIASTASES UPON VENOMS.

Venoms are modified or destroyed by certain normal diastases of the organism. It was shown long ago by Lacerda, Weir Mitchell, Sir Joseph Fayrer, and Lauder Brunton, that it is possible to introduce without danger into the stomachs of adult animals amounts of venom many times greater than the lethal dose. I have repeatedly verified this, but have nevertheless observed that young mammals, while being suckled, readily absorb venom by their alimentary canal, and succumb to the ingestion of doses scarcely larger than those which kill when subcutaneously injected. Here we have a very important fact, which once more proves how easily the intestinal mucous membrane of young animals is permeated by toxins. By my instructions Wehrmann[64] and Carrière,[65] in my laboratory, have studied the modifications undergone by venoms in the alimentary canal of rabbits. We have seen that these animals can ingest without inconvenience doses of venom 600 times greater than the lethal dose, and that, if we cause these ingestions to be repeated several times, contrary to the assertion of Fraser[66] (of Edinburgh), we never succeed in obtaining immunity to the subcutaneous injection of a simple lethal dose, and no antitoxin is formed in the blood.

The _ptyalin_ of the saliva, _pancreatic juice_, and _bile_ destroy _Cobra_-venom _in vitro_. We must, therefore, assume that these diastases are veritable agents of destruction for ingested venom. The intestinal microbes play no part, any more than the intestinal juice by itself. The gastric juice has very little effect. Papain is almost as active as the pancreatic juice.

It had already been proved by Fraser, so long ago as 1895, that bile, after prolonged contact and in a sufficient dose, has a strongly destructive effect upon Cobra-venom; but, contrary to the opinion of this investigator, it is not antitoxic, for it possesses no preventive or curative property, and its effects are produced only _in vitro_.

We see from what has been stated above, that venoms introduced into a sensitive organism are capable of producing extremely complex effects upon the various tissues or humours. They act on the nerve-cells by their _neurotoxin_, on the endothelium of the blood-vessels by their _hæmorrhagin_ (Flexner and Noguchi), on the red corpuscles by their _hæmolysin_, on the fibrin of the blood and muscles by their _proteolytic diastase_, and on the fibrin-ferment itself by their _thrombase_.

They also act on the leucocytes, according to the experiments of Chatenay,[67] performed under the direction of Metschnikoff, and according to those of Flexner and Noguchi,[68] already cited.

Thus we understand how complex must likewise be the means of defence that have to be employed in order to afford an effective protection against such poisons.

The slightly intoxicated organism at first reacts by the intervention of the leucocytes; a _hyperleucocytosis_ is produced, accompanied by a more or less considerable rise of temperature. After a few hours everything returns to its normal condition, and if the injection of a lethal dose of venom is repeated several times, at intervals of a few days, it is not long before antitoxic substances are found to appear in the serum.

When the dose of venom injected is sufficient to cause death, we observe, a very few moments after the injection, a _lowering of temperature_ and a _hypoleucocytosis_, which is the more pronounced in proportion to the nearness of the dose of venom to the minimal lethal dose. With very strong doses the hypoleucocytosis has not time to manifest itself.

It is therefore probable that, in intoxication by venoms as in that by the toxins of micro-organisms, the protective _rôle_ of the leucocytes is all-important, not only because these cells are capable of digesting venoms owing to their protoplasmic digestive juices, but also because they constitute if not the only, at any rate the principal source of the _antitoxic substances_ or _amboceptors_.