Protozoa as Toxicological Tools

The Journal of Protozoology Volume 11 February, 1964 Number I

J. PROTOZOOL. l l ‘ l ) , 1-6 (1964).

Protozoa as Toxicological Tools*

S. H . HUTNER

Haskins Laboratories, 305 E . 43rd St., N e w York 17, N . Y

At the age of 50 a Puka-Pukan retires from manual labor and becomes one of the members of The Company, spending his evenings shooting popguns or playing checkers, and his nights in ordering the young fry about. He never again has to go fishing, to gather coconuts, or to do any other work.

When the malos [young men] bring in their fish they are given to the fathers, who divide them after picking out the choice bits for themselves. . . To be sure, many of the fathers spend a day or two at fishing now and then, but this is not required of them and they do it only to pass the time. . . ( 2 2 ) .

HE experiments sketched here-many by my T younger colleagues-mold shotgun shells from the pharmacopoeia and pop them a t protozoa in the hope that hits will reveal something about protozoan metabolism. The purpose is really phylogenetic: to trace forerunners of the coordination devices that made possible metazoa. Which of those devices originated in the protozoa? One assumes that every device for intercellular coordination began intracellularly. A chemotherapeutic agent that injures protozoa and metazoa but not bacteria hints at a protozoan origin for the metabolic device in question; likewise an antibiotic such as penicillin which kills bacteria but not protozoa or metazoa points to devices confined to bacteria-in the case of penicillin, a kind of cell wall seen only in bacteria and blue-green algae ( 1 1 ) .

Rachel Carson intimates that we are all becoming toxicologists. An upset in the food chain in a Wino- gradsky column-perhaps best one upsetting the protozoa on top-may detect efficiently the chronic toxicity of biodegradation-resistant compounds (29). We will deal here instead with frankly toxic compounds or with the obvious toxicity, a t high concentrations, of compounds ordinarily only moderately toxic. And with carcinogenic hydrocarbons one deals with compounds whose toxicity to protozoa can be increased 1000-fold or more by ultraviolet light, so providing an ultra-sensitive test for these carcinogens.

One aim is to apply the resemblances between protozoa and metazoa to detecting the metabolic sites of

* Address of Past President, Society of Protozoologists, de- livered at Amherst, Mass., 30 August 1963. Much of the research at Haskins Laboratories summarized here was aided by the National Institutes of Health (grants GM 09103 and B-2651).

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damage induced by the by-products of industrialization and urbanization and by the drugs spawned by the technological revolution. That is, we are exploring the relevance of protozoa for detecting the cellular sites of “drug-induced diseases” or “diseases of medical progress.”

Our ventures into micro-pharmacology were encour- aged by the success of the Ochromonas malhamensis Blz assay which completely parallels the metazoan BE metabolic pattern. The Euglena B12 assay is clinically practical as shown by its world-wide acceptance, but the Euglena Blz pattern is very different from man’s. Indeed, as in mammals, the B12 requirement of 0. maZ- hamensis was exaggerated by thyroactive materials (6) , but not euglena’s Blz requirement. Inasmuch as E . gracilis, for all its heterotrophy, has a very restricted permeability-on the order of ChloreZla’s( 3 1 )-the ad- vent of the exceptionally hardy Ochromonas danica was welcome; moreover, 0. danica has the simplest requirements of any known animal, and unlike 0. malhamensis, has a vigorous photosynthesis. Its metazoan- like permeability, as inferred from its sensitivity to a wide variety of antimetabolites, makes it a versatile tool for mode-of-toxicity studies.

The mechanism of a chemotherapeutic agent is how the parasite is selectively damaged (24). The problem thus becomes one of detecting relevant metabolic differences between host and parasite. Administration of a drug together with its metabolic target should cancel effect of the drug, yet administration of folinic acid together with pyrimethamine + sulfadiazine did not suppress their effectiveness in infections with Toxo- plasma gondii but did overcome toxic side effects(Z5). Evidently, differences between host and parasite in

1

2 PROTOZOA AS TOXICOLOGICAL TOOLS

utilization of the metabolite which corresponds to the cellular target of a drug can give rise to therapeutic differentials. All this enhances the urgency in identi- fying the reasons for toxic “side-actions” of a chemotherapeutic or pharmacological agent. Perhaps more commonly such a differential would be lacking. as illus- trated by Jaffe(33) for Trypanosoma equiperdum infections in mice: the reproduction-arresting effect of 6-azauracil was enhanced when infected mice were fed a diet lacking in purines and pyrimidines and conversely, chemotherapeusis by azauracil was partly can- celled when uracil supplemented the diet.

These toxicological studies can be pursued in eat- one’s-cake-and-have-it spirit: If a compound toxic to metazoa is not toxic to protozoa, the situation reflects a metabolic advance in metazoa. If the compound is antiprotozoal. then experiments with protozoa may show how to alleviate toxic side-actions without can- celling therapeutic activity. The uninteresting compounds are those with a uniform cytotosicity from bacteria and blue-green algae to mammalian cells.

PROTOZOA I S S C R E E S I S G FOR ASTITUhlOR COMPOUSDS

Although by definition cancer is a disorganization of multicellular, not unicellular organisms, resemblances between protozoa and metazoa have inspired use of protozoa in screening for antitumor compounds, especially to detect cytotoxic compounds, for most antitumor substances are chronically toxic( 58) , di- rectly cytotoxic( 10). and toxic to microorganisms( 49).

In mammalian tests on new drugs, some 19 distinct observations can be made to define toxicity in respect to organ systems( 53). Information on protozoa might apply to cellular sites of damage. I n comparing microorganisms and transplanted tumors for screening 2 0 0 potential antitumor agents. the media used were those developed as assay media for various metabolites, facilitating detection of specific antimetabolite effects ( 19). Tetrahymena pyriformis. the only protozoan used, had the highest sensitivity of the systems tested, which included 3 tumors( 18). These workers concluded ‘‘ . . . the best use of experimental tumor systems may lie in the secondary and detailed study of those active compounds turned up by the simpler and more rapid primary in vitro systems”( 18).

Since many cytotoxins have metabolic targets represented by thermostable compounds in the peptones. natural extracts. and the like in “p rac t i~a l ’~ media for protozoa. greater sensitivity presumably can be ob- tained by use of minimal media. IIinimal media differ from those devised for conventional microbiological assays: instead of being nutrient-rich, such media must have minimal concentrations of each nutrient. yet the whole medium must support reasonably good

growth. Devising minimal defined media is a tedious task not yet completed for any protozoan let alone for other non-protozoans with comparably complicated requirements. One wonders, therefore, whether a comparison of defined vs. the complex media used by ffest et aL(60) and Johnson et aZ.(34) might have led to a clearer decision as to the advantages of protozoa over bacteria and mammalian cell cultures. In a study(43) in which HeLa cells proved more sensitive to 35 tumor-active antibiotics than (in order of de- creasing effectiveness) Tetrahymena pyriformis, Ochromonas malhamensis, and Crithidia fasciculata, nonetheless the only in vitro activity displayed by several fermentation beers with antitumor activity was against protozoa, hence it was concluded that protozoa are valuable in screening beers. Samuels & Stouder(46) point out that plate tests with strips enable one to detect complex interactions between inhibitors and target compounds.

CARCINOGENESIS AND CYTOTOXINS

And in the winter time the kindly-eyed and reberend old men will sit in front of open fireplaces, with their feet in buckets of hot water, and drink bourbon whiskey with hoarhound candy in it, and think and think and think; and in the summer time they will sit on the front porches, with gin fizzes beside them, and speculate and speculate and speculate (40).

Polycyclic hydrocarbon carcinogens. But protozoology obtrudes on this temperate-zone idyll. Open fires are being outlawed in cities. The paramecium test for ultraviolet-absorbing, polycyclic carcinogenic hydrocarbons( 15,28) seems to parallel the benzpyrene units (benzpyrene being the reference compound for such hydrocarbons) yielded by fire, whether from one cigarette puff or the fires of a power plant. With ben- zene extracts of air filtrates, correlation between paramecium units and benzpyrene units determined by chemical analysis seems so good( 16) that chambers of commerce may yet boast of low-paramecium-unit air as they do now about low ragweed pollen counts or abundance of sunny days. Why live in London- 320 pg benzpyrene inhaled per person per year or Birmingham, Alabama (150 pg)-if one can live in San Francisco (14 pg) while smoking one cigarette pack a day (60 pg) (48) ? “TO blow one’s stack7,-- the practice of blowing soot out of a chimney with a blast of compressed air or steam, generally a t night when no one is looking-may become a lexicographical antique once smokeless fuels, efficient combustion chambers, and precipitrons come into universal use alongside nuclear power plants: the buckets of water alluded to earlier have to be hotted somehow; moreover, air-conditioning (as for those porches) in many localities, e.g., New York City, sets up the peak de- mand for electric power. But atomic power plants

PROTOZOA AS TOXICOLOGICAL TOOLS 3

create new carcinogenic hazards, perhaps mediated by radiomimetic compounds such as 4-nitroquinoline A7-oxide (13) discussed later.

Hepatotoxicity; bourbon and hoarhound candy. My colleague Dr. Herman Baker says it isn’t alcohol as such, rather the “empty calories” represented by su- crose and ethanol that may make liver cirrhosis as often strike soft-drink addicts as alcoholics. Assess- ment of vitamin status and associated liver function will increasingly depend on protozoan assays for such indices as thiamine, nicotinic acid, vitamins B6 and BIZ, and biotin(7). Folic acid and riboflavin may be added when Tetrahymena assays are improved, likewise choline when the choline-requiring tetrahymenas discovered by Elliott et a1.(12) will have been scrutinized,

Protozoa may have a special value in detecting drugs harmless to the infant and adult but harmful to the fetus or the individual with a damaged liver. Presumably the fetus or cirrhotic liver has defective detoxication mechanisms. Presumably, too, protozoa have few if any of the known detoxication mechanisms. We therefore studied thalidomide upon hearing of its teratogenicity. We had already worked with an anti- convulsant, primidone (Fig. 1)) widely used in epi-

Ii 0

Fig. 1. Primidone

lepsy, and used Crithidia to show that its occasional induction of megaloblastic anemia resided in its steric resemblance to folic acid among other heterocycles ( 5 ) . Some barbiturates, eg., phenobarbitone (Fig. 2 ) ,

HN--GO G2H5 / o=c 1 A

HN-CO C6H5 Fig. 2 . Phenobarbitone

also occasionally induce a folic megaloblastic anemia. While thalidomide (Fig. 3) structurally resembles

n

0 H Fig. 3. Thalidomide

barbiturates, we looked for damage other than in folic- acid metabolism although direct folk deficiency or one

4 0

Fig. 4. 4-Nitroquincline N-oxide

induced by administration of antifolics, may be teratogenic also. Trial of thalidomide, several of its breakdown products, and related compounds against our most sensitive test organism, Ochromonas danica, showed that in general the phthalimide moiety inter- fered with nicotinic acid, and the glutarimide moiety with glutamine(20) ; an interference with vitamin K had earlier been noted(21) but this latter seems rather nonspecific, induced by many quinonoid molecules. Since some anti-nicotinics are teratogenic for chick embryos (36) anti-nicotinic effects with protozoa may signify something for metazoa. Data on protozoa tell nothing yet about the conspicuous pharmacological action of thalidomide: sedation. Protozoan data do suggest that structures likely to antagonize nicotinic acid or glutamine are teratogenically suspect-that protection experiments in susceptible animals are in order before large-scale use. That thalidomide may be only one of a family of teratogens is suggested by the activity of d,Z-3-phthalimidoglutarimide and N- phthalyl-d,l-aspartimide (40). As for the gin fizzes, some of the terpenoids in Juniperus berries are likely to be mildly hepatotoxic, which points up the need for more information on hepatic detoxication and other areas of clinical research in which assays by means of protozoa are applicable (7).

The hepatotoxicity of the legendary fusel oils of bourbon seems unknown. Classically these fusel oils are higher alcohols derived from amino acids during fermentation. Wise et aZ.( 62) discovered that high thiamine protects against ethanol, n-propanol, n-butanol, and n-pentanol; e.g., n-butanol 1.2% supports good growth of Polytomella caeca in the presence of 0.02 % thiamine.

4-Nitroquinoline N-oxide ( N Q O ) , carcinogenesis, and tryptophan. NQO (Fig. 4) is a water-soluble, moderately stable, acutely cytotoxic, powerful carcinogen whose electronic configuration may imitate the o-hydroquinones and quinones to which organisms may convert hydrocarbons, carcinogenic or otherwise, much as they do antimalarials(4). Since a paper is in press(65) on how NQO inhibits the growth of several flagellates and bacteria, and more recent work was presented here(64)) only a few points are men- tioned. We began studying NQO because a t low concentrations it halted chlorophyll synthesis in Euglena gradis . If it induced permanently bleached strains en masse it could provide insight into how some flagellates


became colorless. While trying to broaden the very narrow zone between bleaching (temporary and per- manent both) and killing, we found that XQO toxicity was competitively antagonized over a wide range by L-tryptophan. This, coupled with the protection af- forded by tryptophan against photodynamic killing of paramecium( 14) suggested that a condition for car- cinogenicity, a t least in the hydrocarbon series, is deception of the tryptophan-transport system( 65). Tryptophan protection is being studied( 64) : reducing agents also protect ( 14).

An unpublished study (R. &I. Smillie, A. C. Zahalsky, and M. Keane) with Euglena gracilis shows that SQO stops electron transfer probably between cytochrome b and f , perhaps where nonyl-hydroxyquinoline S- oxide does. The nitro group in NQO probably confers a strong uncoupling of oxidative phosphorylation. This ties carcinogenesis to interruption of respiration -a reminder of Otto TVarburg’s much-discussed thesis [e.g., by Gause(22) in relation to finding antitumor substances by means of microorganisms with impaired respiration that carcinogenesis is related to impaired respiration.

I t would be premature to urge that ingestion of bourbon, gin or candy, or inhalation of benzpyrene- laden air, be accompanied by a canapk fortified with tryptophan, choline, methionine. etc., along with a vitamin pill high in vitamin E and thiamine.

A?tIISOC I’CLOALKASES

Use of protozoa to discern mode of toxicity (if not mode of action in the G. H. Hitchings sense) is illus- trated by the aminocycloalkanes. The report that 1 -aminocyclopentane- 1 -carboxylic acid ( ACP) (Fig. 5 ) acted against multiple myeloma and some animal tumors, and that its mode of toxicity was unknown ( 8 ) , inspired a study of the toxicity of ACP towards Ochromonas danica. Toxicity was annulled by L- alanine and glycine in that order, and inhibition by 1-amino-3-methylcyclohexane-1-carboxylic acid (AMCH) was annulled by L-leucine( 1). These results may be compared with ( a ) the effectiveness of valine in alleviating the toxicity of ACP in chicken diets--a toxicity intensified by leucine(37) : and ( b ) noncompetitive inhibition in ascites cells of glycine uptake and competitive inhibition of valine and leucine transport ( 54).

0. danica did, then. point to the relevant metabolic pathways in vertebrates.

\ / N H z C-C

! c / ‘ C O O ” c-c

Fig. 5 . 1-.4minocyclopentane-I-carboxylic acid

PROTOZOA AND ANTIHORMONES

A big gap in comparative pharmacology, as in bio- chemistry generally, is that represented by the lower metazoa. A recent survey(l7) indicates that in the mollusc-annelid-arthropod line, acetylcholine and adrenaline act much as they do in vertebrates, but with differences; thus although the clam (Venus mer- cenaria) heart is very sensitive to inhibition by acetylcholine. atropine does not usually block the effect. Histamine responses are irregular in the invertebrates tested so far. Coelenterates do not respond regularly to acetylcholine. Consequently it is unsurprising that neuro-effectors do not affect protozoa in textbook style. Thus the rule of acetylcholine in flagellar and ciliary movement is a much-disputed issue; Parame- cium, Euglena and Ochromonas are insensitive to physostigmine and neostigmine( 2 ) , likewise physostigmine for Tetrahymena( 56) ; immobilization of Tetrahymelza by 3 x lo-? M hexamethonium C1 was not reversed by acetylcholine analogs. But acetylcholine and anticholinesterases did make the negative galvanotaxis of Paramecium disappear or replaced it by a positive galvanotaxis( 3 5 ) . Again, immobilization of Tetrahymena and Ochromonas by several antihistamines was counteracted by histidine, not histamine( 44). Equally puzzling is bleaching by streptomycin-type antibiotics. It is so consistent(63) as to suggest that new antibiotics of this type should be scrutinized for damage to the 8th cranial nerve which controls hearing and balance if they bleach Euglena, and that, conversely, should such an antibiotic not bleach. it might be the long-sought non-ototoxic streptomycin-type antibiotic. (One wonders whether a streptomycin-bleached euglena swims upside down ! )

Since all antihistamines have toxic “side actions” ( 5 1 ), and the antihistamines overlap with many other classes of drugs, including the phenazine tranquilizers now being used on a tremendous scale a t high doses over long periods of time, studies of the toxicity of these drugs, even a t “unphysiologically high” levels, is warranted. We have also studied the toxicity at high concentrations of some of the radio-opaque agents used in radiographic studies which are also very widely used; our results(39) are encouraging in that inhibition of Tetrahymena motility parallels toxicity to mammals but the technique is far from quantitative; far more work remains to be done.

.-lntisterols. One way to see whether protozoa have steroid hormones is to expose them to compounds blocking one or another step in sterol metabolism and see whether sterols and steroid hormones counteract any growth inhibition. If steroid hormone synthesis were blocked at the critical elimination of the aliphatic side chain, and such hormones were necessary, steroid hormones should counteract the growth inhibition. In

PROTOZOA AS TOXICOLOGICAL TOOLS 5

this sense the experiments so far have failed(30) ; thus tripanol inhibition of Ochromonas danica was annulled by fatty acids(3) ; triparanol inhibition of Tetrahymena pyri f ormis was likewise annulled by fatty acids, with sterols acting additively( 26). The block in fatty-acid synthesis, because of the effectiveness of oleate, could be the unsaturation step. Perhaps there is a comparable block in unsaturation of a double bond in the sterol molecule. The principal non- saponifiable lipid in Tetrahymena is a triterpenoid, not a stero1(38), which uncovers some fresh bio- chemistry.

MISCELLANEOUS POISONS

The presence of “cytoplasmic” poisons in high concentrations in higher plants, notably colchicine and the quinine alkaloids, bespeaks cytoplasmic compart- mentation or a non-metazoan-like metabolism. The same puzzle confronts one with the alkaloid-like dino- flagellate toxin (50) : What is this toxin doing in the flagellate? Why are dinoflagellates immune to i t? Consistent with the apparent inability of this and other neuro-affector agents to affect protozoa in metazoan fashion is the indifference of Tetrahymena to staphylococcal toxins (9) ; also one awaits confirma- tion with axenic cultures of the report that tetanus toxin paralyzes Paramecium azcrelia (45).

Urethan. Growth inhibition of ~ o ~ e r i o c h y o ~ z o ~ a s stipitata by urethan is counteracted by thymine(32). In contrast, urethan inhibition of Escherichia coli is annulled by phenylalanine + glu tamic acid (6 1 ) or adenine( 5 2 ) . Since urethan-resistant E. coli is resistant to formamide and N-methylformamide, protection by thymine against toxicity to rats of the related dimethylformamide, though not against N-methylformamide ( 5 5 ) , is provocative. Formamide, N - methylformamide, and N-methylacetamide are missed by all in vitro screens for antitumor agents( 18,19).

Antimalarials. Some alluring byways are revealed among antimalarials. Chloroquine is widely used for treating rheumatoid arthritis, and quinidine is used for checking auricular fibrillation. The puzzlement a t inability of quinacrine (atabrine) to affect tumor growth(24) finds some resolution in the report that weighting quinacrine-type acridines by Br substitu- tion yields tumor-active compounds (44) . Quinacrine is useful for local killing of tumor cells(57). Quini- dine and quinine are remarkably anabolic without being androgenic(27). We need to know how antimalarials affect other metabolites besides such obvious targets as flavins and folic acid.

Before yielding to pessimism about the prospects of eliminating toxicity without simultaneously eliminating therapeusis, one may recall that until the acridine antimalarials came along there was no reason to sup-

pose that ototoxicity, as displayed by the quinine alkaloids, was dissociable from antimalarial activity.

CONCLUSION

In laying before you these selections from our catch, I hope you will agree that the nascent subject of toxi- cologicaI protozoology, despite its conspicuous limita- tions, has a sunny future.

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Protozoa as Toxicological Tools