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The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity

School of Biochemistry and Molecular Genetics and Cellular Analysis Facility, School of Microbiology and Immunology, University of New South Wales, Kensington, Sydney 2052, Australia¹
Microbiology Group, School of Biosciences (BIOSI, Main Building), Cardiff University, Cardiff CF10 3TL, UK²

Author for correspondence: David Lloyd. Tel: +44 29 2087 4772. Fax: +44 29 2087 4305. e-mail: lloydd{at}cf.ac.uk

Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O₂. Thus, when exposed to controlled concentrations of dissolved O₂ >8 µM, they gradually lose their ability to scavenge O₂. In a washed cell suspension stirred under 10% air in N₂ (equivalent to 25 µM O₂), inactivation of the O₂-consuming system was complete after 3·5 h; during this period accumulation of H₂O₂ (3 µmol per 10⁶ organisms) and oxidation of cellular thiols to 16% of their initial level occurred. Under 20% air (50 µM O₂), respiratory inactivation was complete after 1·5 h, and under air (258 µM O₂), after 50 min. Loss of O₂-consuming capacity was accompanied by loss of motility. Use of the fluorogen 2,7-dichlorodihydrofluorescein acetate indicated that intracellular H₂O₂ is produced at extranuclear sites. Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC₄(3), showed that values declined from -134 mV to -20 mV after 4·5 h aeration. Incubation of organisms with 60 µM H₂O₂ for 10 min gave partial collapse of plasma membrane potential and complete loss of O₂ uptake capacity; motility and viability as assessed by DiBAC₄(3) exclusion were completely lost after 1 h. Inactivation of the O₂-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.

Keywords: hydrogen peroxide, oxidative stress, reactive oxygen species

Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N’-dicyclohexylcarbodiimide; DiBAC₄(3), bis(1,3-dibutylbarbituric acid) trimethine oxonol

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