trans

ACTION OF NITROIMIDAZOLE DRUGS 135
levels in the medium correlate positively with
levels in the hydrogenosomal proteins in this
species. Interestingly, proteins without iron–
sulfur centers were also affected, indicating
that this effect is not due simply to the availability
of iron for incorporation into iron–sulfur
proteins.
Genetic transformation with dsRNA Giardia
virus as vector has been used to affect the
levels of PFOR and alcohol dehydrogenases
in G. intestinalis. The results confirmed the
role of PFOR in metronidazole activation, and
showed that of the several alcohol dehydrogenases
present in this organism only the fused
aldehyde/alcohol dehydrogenase plays a significant
role.
ACTION OF
NITROIMIDAZOLE DRUGS
The most widely used selective drugs against
amitochondriate parasitic protists are nitroimidazole
derivatives (Chapter 17). Metronidazole
(1-hydroxyethyl, 2-methyl, 5-nitroimidazole)
was the first member of this group to be introduced
and remains a much used compound.
The NO 2 group has to be reduced to convert
these compounds into cytotoxic agents. The
reduction is driven by low redox-potential electrons
generated from pyruvate oxidation by
pyruvate:ferredoxin oxidoreductase and passed
on to ferredoxin. Other redox enzymes have
been proposed as additional electron sources.
Ferredoxin is generally assumed to be the proximal
electron donor in the reduction of
5-nitroimidazoles. Inhibition of H 2 formation
in T. vaginalis by nitroimidazole shows that the
drug competes with hydrogenase for the electrons.
The products of the reduction are
short-lived cytotoxic products, and their exact
identity remains uncertain despite extensive
research. The one-electron product, nitro free
radical, and the two-electron product, a hydroxylamine
derivative, have been proposed as
active compounds.
If oxygen is present, the level of metronidazole
toxicity decreases. It is unclear whether
this effect is due to diversion of the electrons
to oxygen as an alternative acceptor or to the
reoxidation of the one-electron free radical.
The reoxidation of the free radical leads to the
generation of reactive oxygen species (e.g.
superoxide). The contribution of such secondary
toxic species to the cytotoxicity of the drugs
is not known. It is significant that 5-imidazoles
exert their anti-parasitic activity also under
strictly anaerobic conditions.
Relative 5-nitroimidazole resistance has
been observed for the three human pathogens.
Resistance can also be developed in cultured
organisms exposed to increasing concentrations
of the drugs. The levels of resistance seen
in clinical cases and those obtainable under
laboratory conditions are species-dependent.
Moderate resistance can be easily obtained in
T. vaginalis, less easily in G. intestinalis and
only with great difficulty in E. histolytica.
The mechanism of resistance can often be
attributed to decreased levels of components of
the system reducing the drug. In low, clinically
relevant, levels of resistance in T. vaginalis, the
reducing system is unchanged, but the organisms
are less oxygen-tolerant, indicating that
modifications to the oxygen detoxifying system
affect the toxicity. In laboratory strains of
G. intestinalis with decreased drug susceptibility,
decreases in the reducing system are accompanied
by increased levels of enzymes involved in
oxygen detoxification.
Long-term (over a year) cultivation in the
presence of increasing metronidazole levels
of both trichomonad species can achieve
extremely high tolerance to the drug. During
the process the organisms gradually lose their
PFOR and hydrogenase activities and finally are
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA