The Effect of Metronidazole and Nitrofurans on the Morphology of Trichomonas Vaginalis

Journal of Antimicrobial Chemotherapy (1975) 1,229-234

The effect of metronidazole and nitrofurans on the morphology of Trichomonas vaginalis

Yvonne Bnchner and D. I. Edwards* Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021

Drug Research Unit, Department of Applied Biology, North East London Polytechnic, Romford Road, Stratford, London El 5 4LZ, England

Metronidazole, a nitroimidazole, and the nitrofurans nitrofurazone, nitrofurantoin and SQ 18506 caused extensive cytoplasmic vacuolation in Trichomonas vaginalis cells with possible effects on the nuclear membrane. The cytoplasmic effects may be due to the disruption of chromatic granules or hydrogenosomes. SQ 18506 and nitrofurazone also caused electron dense condensation of nuclear chromatin. It is postulated that the cytological effects of the drugs are correlated with their effects on the pyruvate phosphoroclastic system of Trichomonas vaginalis

Introduction Nitrofurans, e.g. nitrofurazone, nitrofurantoin, have been used clinically as antibacterial drugs for over 25 years, mainly for the treatment of urinary tract disease. The nitrofuran SQ 18506 has trichomonacidal, antibacterial, antimonilial and antibilharziaJ activity (Gadebusch & Bach, 1974). Its trichomonacidal activity is similar to metronidazole (a nitroimidazole) which is currently used for trichomoniasis, intestinal amoebiasis and Vincent's disease. The effect of metronidazole on the morphology of the protozoan pathogen Trichomonas vaginalis has been studied using electron microscopy by Perju, Petrea & Toader (1963) and Panaitescu, Voicultescu, Ionescu & Petrovici (1970) with widely differing and inconclusive results. Perju concluded that "cytotoxic activity predominated on the microgranular systems, and the fundamental plasma of the organism", while Panaitescu and others thought the drug's prime effect was to cause "degenerative lesions of the nucleus". In order to clarify the contradictory conclusions of previous workers, and to obtain further evidence for the mechanism of action of metronidazole and also nitrofurazone, nitrofurantoin, and SQ 18506, we investigated their effect on the morphology of T. vaginalis using Nomarski differential interference contrast microscopy (Nomarski, 1955) and electron microscopy. Structures of the compounds used in this study are shown in Figure 1. • Requests for reprints should be addressed to D. I. Edwards. 229 230 Y. Bncfaner and D. L Edwards

Methods and materials Organism One strain of Trichomonas vaginalis from a clinical sample was obtained by courtesy of Dr R. Simons from the Middlesex Hospital, London and maintained in Bushby's medium (Bushby & Copp, 1955) at 37°C.

Metronidazole Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021

OZN CH2CH2OH

(I - $ - hydroxyethyl - 2 - methyl - 5 - nitro - imidazole)

Nitrofurazone

02N ^CT ^CH= N-NH-CO-NH2

( 5-nitrofurfuraldohyde semicorbazone)

Nltrofurantoin

(l-(5-nitrofurfurylidene omino) hydarrtoin)

SO. 16506

Figure 1. Structure of compounds used in this study.

Compounds Nitrofurantoin, nitrofurazone, and SQ 18506 were dissolved in a minimum volume of dimethylfonnamide (DMF) and made up to the required concentration in sterile 0-85% (w/v) aqueous sodium chloride. Metronidazole was dissolved in sterile 0-85% (w/v) aqueous sodium chloride. The final concentration of DMF was about 002 % (v/v) and had no effect on the viability of T. vaginalis. Solutions were sterilized by membrane filtration (pore size 0-2 u). Effect of drags on T. vaginalis morphology 231

Assessment of viability Drugs were added to the organism in Bushby's medium and viability was assessed as previously described (Edwards & Mathison, 1970). Minimum inhibitory concentration (MIQ determinations were carried out using doubling dilutions to 0-1 ug/ml and assessing viability at 24 and 48 h. The highest dilution in which there was no movement of flagella or undulating membrane and no growth at 48 h was taken as the MIC.

Microscopy The four drugs were added to T. vaginalis (growing exponentially) at concentrations of 200 times the MIC. Samples were removed at 5-min intervals from 0 to 30 min, 10-min intervals from 30 to 60 min, and at 80, 100 and 120 min. Control samples were removed Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021 at the time indicated from cultures containing no drugs. For light microscopy wet preparations were made by placing 1 drop (0 02 ml) of culture on to warmed (37°C) slides, covering with a no. 1 ± coverslip and sealing with molten paraffin wax. These preparations were photographed using Nomarski differential interference contrast microscopy through a XI00 oil immersion lens with an Optovar magnification of 1 -25. For electron microscopy samples were immediately placed in 3% glutaraldehyde buffered with 0-1 M-sodium cacodylate pH 7-2, for 2 h. The organisms were then centrifuged at 627 g for 5 min, washed in Tyrode solution and post-fixed for 1 h in 1 % osmium tetroxide in cacodylate buffer containing 0-2 M-sucrose. Further centrifugation in Tyrode solution was carried out 3 times and the organisms re-suspended in Tyrode solution for 4 h. The packed cells were placed by means of a fine spatula on the surface of dental wax and cut into blocks. These were dehydrated in increasing concentrations of ethanol to 100 % and embedded in Spurr's resin (Spurr, 1969). Sections were mounted on collodion-coated grids and stained with 2 % aqueous uranyl acetate followed by lead acetate (Reynolds, 1963). They were examined on a Siemens Elmiskop 1A microscope using a 50 urn objective aperture at a magnification of 5000 at 80 kV.

Results The MIC values of the three nitrofurans and metronidazole against T. vaginalis are shown in Table I. All drugs has a similar effect on the organism as adduced by light microscopy, killing the cells between 40 and 50 min and producing cytoplasmic extrusions or protrusions concomitant with rounding of the cell and subsequent lysis. Metronidazole and SQ 18506

Table L MIC values for three nitrofurans and metronidazole against Trichomonas vaginalis

Trichomonas vaginalis Compound MIC Metronidazole 1-0 Nitrofurazone 3-2 Nitrofurantoin 3-2 SQ 18506 6-4

All values are given in ug/ml, and are averages of duplicate experiments. 232 Y. Bnchner and D. I. Edwards

caused extrusions at 15 min which became more extensive to 120 min. With nitrofurantoin extrusions first appeared at 20 min and with nitrofurazone at 30 min (Plate 1). In the electron microscope cells exposed to SQ 18506 showed cytoplasmic deformations at 10 min (5 min before changes were observed in the light microscope). The main effect was an apparent reduction in the number of chromatic granules and the appearance of apparently empty vacuoles. At 50 min the vacuoles were large and they increased in size to 120 min when they caused breaks in the cell membrane. After 30-min condensation of electron dense material with the nuclear membrane occurred with breakdown of the membrane; this was complete at 60 min when no nuclear membrane was observed The effect of metronidazole was similar to SQ 18506 but no nuclear condensation occurred (Plate 2).

With nitrofurantoin cytoplasmic deformations were first noticed at 15 min with vacuola- Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021 tion becoming extensive from 50 to 120 min. At 80 min about 7C % of the cell was composed of these vacuoles, and at 120 min they were causing membrane breaks resulting in lysis. There was no obvious effect on the nuclear organization although in some specimens the staining ability of the nuclear membrane was poor. Nitrofurazone caused cytoplasmic disruption at 15 min, the cells becoming extensively vacuolated at 50 min. Between 20 to 40 min electron dense condensations appeared in the nucleus, but these disappeared by 50 min. At 100 min the vacuoles were large, causing breaks in the cytoplasmic membrane and lysis. The integrity of the nuclear membrane appeared to be affected in some specimens.

Discussion Some workers consider the chromatic granules in Trichomonads to be equivalent to mitochondria (Joyon, 1963; Nielsen, Ludvik & Nielsen 1966; Nielsen, 1972). Others state that they occur with and are distinct from mitochondria (Anderson & Beams 1959, 1961; Chakraborty, 1961; Sharma & Bourne 1963; Yeh, Huang & Lien, 1966); while others claim that as the cells are anaerobic mitochondria are absent and therefore the chromatic granules must be a distinct organelle (Smith & Stewart, 1966; Honigberg, Mattern & Daniel, 1971; Daniel, 1972; Muller, 1973; Lindmark & Muller, 1973). Recent and most convincing evidence comes from Muller (1973) and Lindmark & Muller (1973) who have demonstrated in T. foetus that the chromatic granules contain a pyruvate phosphoroclastic system of the clostridial type. They proposed the name "hydrogenosomes" for them and distinguished them from peroxisomes, lysosomes, and mitochondria. Metronidazole and nitrofurazone inhibit either H, (metronidazole) or CO2 evolution (nitrofurazone) from the clostridial-type pyruvate phosphoroclastic system of T. vaginalis (Edwards, Dye & Came, 1973). The morphological effects of these drugs substantiates the work of Muller (1973) and Muller & Lindmark (1973) as their biochemical action appear to be related to morphological changes of the chromatic granules or "hydrogenosomes". The lethal event in the cell may not be the inhibition of the phosphoroclastic system as has been previously suggested (Edwards et ah, 1973), for the antimicrobial activity of nitrofurans is linked with reduction of the nitro group (McCalla, Reuvers & Kaiser, 1970) and reduced nitrofurans inhibit DNA function and cause chromosome breakage in bacteria (McCalla, Reuvers & Kaiser, 1971). It is not surprising, therefore, that SQ 18506 and nitrofurazone affect the nucleus of T. vaginalis, but is it significant that Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021

Plate 1. T. vaginatis photographed using Nomarski differential interference contrast microscopy, (a) Normal cell in absence of drug, (b) After exposure to 640 ug/ml nitrofurantoin for 50 min. Lettering guide to figures A, Axostyle; B, bleb; F, flagellum; H, hydrogenosome; L, lysosome; N, nucleus; PF, posterior flagcllum; UM, undulating membrane. Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021

lum

1um Ca) (b)

Plate 2. Electron micrographs of T. vaginalis. (a) Normal cell in absence of drug, (b) After exposure to 200 ug/ml metronidazole for 50 min. (See legend to Plate 1 for abbreviations). Effect of drags on T. Vaginalis morphology 233 this occurred after cytoplasmic disruption. We infer that the drugs were reduced by cytoplasmic hydrogenosomes and that reduction products initiated the events in the nucleus. Similar events may occur with metronidazole which powerfully inhibit RNA and DNA synthesis in T. vaginalis (Ings, McFadzean & Ormerod, 1974). Although we have not detected morphological changes within the nucleus of cells exposed to the drug the effect may be a subtle one. We suggest that further work to establish a quantitative correlation between drug reduction, phosphoroclastic activity, hydrogenosome morphology and cell viability would be valuable. A parallel study on drug induced morphology of T. vaginalis using scanning electron microscopy is in progress. Downloaded from https://academic.oup.com/jac/article/1/2/229/718197 by guest on 02 October 2021

Acknowledgements We thank May and Baker Ltd., Dagenham, Essex, for supplying metronidazole; Smith Kline and French, Welwyn, Hert., for nitrofurazone and nitrofurantoin, and Squibb and Sons Inc., Princeton, New Jersey, U.S.A. for SQ 18506.

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