MICROBIOLOGICAL REVIEWS, Mar. 1982, p. 86-94 Vol. 46, No. 10146-0749/82/010086-09$02.00/0

Bacterial Toxins: a Table of Lethal AmountsD. MICHAEL GILL

Tufts University School of Medicine, Department of Molecular Biology and Microbiology, Boston,Massachusetts 02111

SUMMARY ..............................................................SELECTION OF DATA AND SOURCES OF ERROR.........................THE TABLE .............................................................DISCUSSION.............................................................

Relevance to Possible Cloning of Genes Coding for Toxins....................Recommended Containment Levels for Cloning .............................

(i) Proteins with an expected 50% lethal dose for humans of 100 ng/kg or less.(ii) Proteins with an expected 50% lethal dose for humans of >100 ng/kg and<1 agfkg ..........................................................

(iii) Proteins with an expected 50% lethal dose for humans of 1 to 100 ag/kg .(iv) Proteins of low toxicity .............................................

Risks Associated with Cloning Toxin Genes in Escherichia coli ................Literature Cited ...........................................................

86869090909191

9191919192

SUMMARYThe amounts of bacterial toxins and of some

plant and animal proteins that kill humans, mon-keys, mice, guinea pigs, and rabbits are tabulat-ed and are discussed in the light of guidelines forthe cloning of genes coding for toxins.

SELECTION OF DATA AND SOURCES OFERROR

The values in Table 1 have been recalculatedfrom the original data to minimize copying er-rors in reviews, but considerable sources oferror remain.Almost always the major problem in interpret-

ing the literature is to establish the purity of thepreparations used and the degree of inactivationsuffered during isolation. An attempt was madeto list toxicity values obtained only from homo-geneous material; except for the toxins that areso abundant in filtrates that purification is sim-ple, this principle entailed the omission of manyvalues obtained before modern methods of pro-tein purification and analysis were available.A few values (for the anthrax toxin complex,

Clostridium perfringens beta-toxin, and Yersiniapestis murine toxin) are included even thoughthe published data do not allow purities of thepreparations to be estimated. These values havebeen placed in brackets and "<" has been usedto emphasize that the figures are maximumvalues. Some even more suspect data have beenrelegated to footnotes, and a few toxins havebeen listed which are probably lethal but forwhich no data have been found. In general,although the literature frequently contains wide-ly different estimates of toxicities, only the mostlethal values are included in the table because

these probably represent the purest material andthe least inactivation. For toxins that requirepartial proteolysis for full expression of activity,values are listed only after activation.The opposite problem of spuriously high po-

tencies arises for proteins of limited toxicitiesthat were isolated from the products of bacteriawhich produce several toxins. Staphylococci,streptococci, and clostridia are examples. Manyproducts prepared from C. perfringens, includ-ing commercial enzymes, are contaminated withthe cytolytic theta-toxin (63).A third source of error is the inherent in-

accuracy of the determinations themselves. Thenumber of animals used to determine toxicitiesis frequently insufficient for the accuracyclaimed, but the offense is usually only a statisti-cal one, for great accuracy is of no merit in thesedeterminations. The amount of toxin required tokill a particular animal is specific for that animalon that occasion and clearly cannot be measuredwith any precision. It can only be estimatedfrom other individual animals whose physiologi-cal conditions may not be the same. A particu-larly severe variation concerns the pyrogenictoxins, the apparent lethalities of which varyover several orders of magnitude according tothe endotoxin load of the test animals.Workers have used a variety of routes of

injection for toxins being tested. When speci-fied, the route is listed in the table. Intravenousinjection is often a few fold more effective thanintraperitoneal injection. Intramuscular or sub-cutaneous injections are often severalfold lesseffective than intravenous ones. Intracranial orintraspinal values are not given, although theseroutes are much more effective for many toxins,both the classical neurotoxins (10) and others.

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Values are expressed per kilogram of bodyweight, assuming, when necessary that the miceweighed 20 g, the guinea pigs weighed 250 g, andthe rabbits weighted 3 kg. Such normalizationimplies a linearity between dose and weight thatprobably holds only rarely. The assumption hasbeen explicitly challenged for botulinum toxinby Lamanna (47).For all of these reasons, the values in the table

must be interpreted carefully. At best they areaccurate to one significant figure. Usually theyare provisional values that may be reviseddownwards and serve now to define only thelikely maximum size of the lethal dose.

THE TABLEMost values are given as 50o lethal dose

(LD50) per kilogram. Those in parentheses areminimum lethal dose (MLD), or LD100, perkilogram. Some authors assume that the MLD isabout twice the LD50, but there is no constantrule. For botulinum toxin, which was titratedwith care, the factor is about 1.6 (47). Fortetanus toxin it is about 1.4 (91). For abrin andricin it is close to 1.0 (33). For the greatestaccuracy the time within which the animals dieshould be specified, but this information is oftenomitted. The exception is the "MLD" of diph-theria toxin, which has a somewhat differentmeaning that includes a time of death (see foot-note h). Values are given as mass of protein,assuming, when necessary, that the protein con-tained 16% N.

In part I the bacterial proteins are arranged inalphabetical order of parental bacterium. Forcomparison, the lethalities of some nonbacterialtoxins are included. Part II lists plant proteintoxins that have been purified and assayed. PartIII is not comprehensive but presents a sampleof the neurotoxic proteins from snake and inver-tebrate venoms. More are listed by Tu (90), butmost venoms have been assayed only as mix-tures and the potencies of the individual com-ponents are unknown. The purified venom neu-rotoxins are nearly always lethal to mice at 10 to100 ,ug/kg. Taipoxin is unusually potent.

DISCUSSIONRelevance to Possible Cloning of Genes Coding

for ToxinsOne reason for compiling these data arose

when the National Institutes of Health Recombi-nant DNA Advisory Committee and its ad hocworking group on toxins considered the dangersthat might develop from the cloning of genes forbacterial and other toxins. The discussions re-sulted in guidelines for cloning toxin genes inEscherichia coli (Fed. Regist. 46:34487, 1 July1981). A novel feature is a recommendation that

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LETHAL AMOUNTS OF BACTERIAL TOXINS 91

different containment levels should be used fortoxins of different lethalities.During our discussions it became apparent

that, whereas the risk to humans would dependon a toxin's toxicity to humans, human datawere not often available and would have to beinferred from values obtained with other ani-mals. Our best recourse would be to extrapolateto humans from measurements on other pri-mates. For this reason, the table includes pub-lished data for monkeys. Unfortunately, formany toxins the only indication of likely humantoxicity comes from experiments with nonpri-mate mammals, most often mice, and experi-ence shows that there is often a very poorcorrespondence between toxicity to humans andthat to any one small animal (e.g., diphtheriatoxin, shigella "neurotoxin"). Nevertheless, weneed some way of predicting human toxicities,and it has been proposed that, unless or untildirect measurements on primates are made andsolely for the purpose of selecting the appropri-ate containment level in a cloning experiment,humans be assumed to be as susceptible to aparticular toxin as the most susceptible of threesmall mammals, mice, guinea pigs, and rabbits.As the table reveals, there are few toxins forwhich adequate small animal data are available,but they would not be hard to collect whennecessary.

Recommended Containment Levels for CloningThe guidelines suggest four classes. The divi-

sions between the classes are, perforce, some-what arbitrary but represent the working group'sbest sense of convenience and prudence, giventhe limited knowledge available. For most toxinsextra data will be required to determine theappropriate class.

(i) Proteins with an expected 50% lethal dosefor human of 100 ng/kg or less. In effect, thismeans 50%o lethal for humans, for monkeys, orfor the most sensitive of mice, guinea pigs, andrabbits. Cloning is prohibited (without specialpermission of the National Institutes of Health).This group presently contains the botulinumtoxins, tetanus toxin, the shigella neurotoxin,and diphtheria toxin. Others might enter thisgroup when more data become available.

(1i) Proteins with an expected 50% lethal dosefor humans of >100 ngfkg and <1 gLg/kg. Cloningis permitted under P2 + EK2 or P3 + EK1containment. Abrin seems likely to belong tothis group, as do ricin, modeccin, C. perfringensepsilon-toxin, and C. difficile enterotoxin.

(ill) Proteins with an expected 50% lethal dosefor humans of 1 to 100 gLg/kg. Cloning is permit-ted at P1 + EK1 containment. Extrapolation tohumans from the small-animal data places strep-tolysin 0 in this class, and it appears likely that

other oxygen-labile hemolysins will belong heretoo, as well as many other toxins, but the dataare usually not sufficient to allow decisions yet.

In addition, cloning of cholera toxin-like andST (heat-stable)-like enterotoxins is permittedunder P1 + EK1 containment, even if theyshould prove to be more potent than 1 ,uag/kg forhumans, for the reasons discussed below.

(iv) Proteins of low toxicity. These proteins,lethal to humans at over 100 ,g/kg, are notsubject to specific restrictions on cloning (ex-cept for the enterotoxins in group 3). An exam-ple is the delta-lysin of Staphylococcus aureus.

Risks Associated with Cloning Toxin Genes inEscherichia coli

These categories and the guidelines apply onlyto cloning in E. coli. The risk inherent in using adifferent host would depend on the habits of thisorganism. It must be emphasized that the habitsof a gene's former host, including its ability tocause disease or to exchange genetic informa-tion, are no longer relevant once the gene istransplanted. For example, the knowledge thatC. tetani exists in the normal bowel withoutpathology does not make it any safer to transferthe gene for tetanus toxin into another intestinalorganism. For E. coli the major risk seems to liein the production of a toxin in the intestine byeither E. coli itself or another intestinal organismthat acquired the toxin gene from E. coli. Wecan imagine three types of dangerous outcomes.

(i) Some of the toxin might pass out of thebowel into the general circulation and damagedistant tissues. This would be most apparent forthose such as tetanus and botulinum toxins,which have no effect on the bowel itself butwhich inactivate neural synapses. That somebotulinum toxin escapes into the circulation isimplicit in every case of botulism: possiblemechanisms have been discussed by Bonventre(19). Only about 1 part in 100,000 of orallyadministered botulinum toxin escapes (47), but agreater proportion might escape if the toxin wereto be made in the gut itself and avoid inactiva-tion by the stomach. Wright (95, p. 420), inreviewing a few experiments in which botulinumtoxin was placed in the ileum, ileal loops, andcolonic loops, concluded, "What slender evi-dence there is available thus suggests that mostof the absorption of these toxins must take placein the stomach or in the upper portions of thesmall intestine." Shigella neurotoxin is substan-tially inactivated in the stomach (20). The riskmust be greater for adults in whom passage ofproteins from the intestine is rendered morelikely by such conditions as ulcers or intestinalrupture or for neonates.

(ii) Many of the toxins that are lethal wheninjected parenterally are cytotoxic and if pro-

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duced in the intestine will presumably causenecrosis and ulceration in the mucosa and con-sequently diarrhea or dysentery. The cytotoxicenterotoxin of Shigella dysenteriae is thought toact thus (41). The mucosal damage might also befollowed by a greater leakage of the toxin intothe circulation, which would pose an additionalrisk.

(iii) The noncytotoxic enterotoxins such ascholera toxin and the heat-stable and heat-labileenterotoxins of E. coli would presumably causesecretion in the same way that they do in thenatural diseases. Despite the fear historicallyassociated with the word cholera, the dehydra-tion consequent on the diarrhea is completelyreversed by oral and intravenous administrationof electrolyte solutions and, given proper care,the risk to an experimenter from a neocholeraorganism may be limited to discomfort.Except for the enterotoxins, there are few

data on the safe amounts of toxins in the guts ofexperimental animals, let alone in humans. Wecan only proceed on the temporary assumptionthat a relation exists between enteral and paren-teral toxicities. We must assume that a toxinwhich kills when minute amounts are adminis-tered to the blood may also be a significantdanger when produced in the gut, and that themore toxic it is, the greater the barriers thatshould be erected to restrict the toxin's produc-tion in the intestine. This may be accomplishedeither by imposing physical containment or byusing strains of E. coli that do not colonize andhave less opportunity to transfer their geneticinformation to abundant intestinal residents.

ACKNOWLEDGMENTS

I thank the following people for their help: John P. Arbuth-nott, Alan W. Bernheimer, Kathy K. Clark, Richard A.Finkelstein, John H. Freer, Neal B. Groman, Elizabeth Mi-lewski, Siur Olsnes, and John Stephen.

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