EFFECT OF ARTESUNATE ON RAT LIVER

MITOCHONDRIAL MEMBRANE PERMEABILITY

TRANSITION PORE OPENING (IN VIVO)
A marked and prolonged increase in Ca2+ is harmful to cells because it leads to activation
of calcium-dependent enzymes such as lipases, propteases, endonucleases and
phosphatases having a potentially adverse effect (Pferiffer et al., 1998). Pferiffer et al.
(1998) hypothesized that the transition reflects a Ca2+-induced increase in phospholipase
A2 activity with lipid degradation products increasing membrane permeability.

Mitochondria modulate the free cytosolic Ca2+ concentration during and following intense
calcium conductances in plasma membrabe (Friel and Tsien, 1994; White and Reynolds,
1996; 1997; Wang and Thanyer, 1996). At steady state, there is a balance between influx
and efflux of Ca2+ across the mitochondrial membrane. Mitochondria start to accumulate
calsium when the cytosolic calcium concentration rises over a threshold (about 500nM).
Ca2+ uptake by the mitochondria occurs via a uniporter (channel), and si driven by the
mitochondrial membrane potential. The rate of uptake is proportional to Ca 2+
concentration. The inward movement of Ca2+ increases the transmembrane potential,
which is restored either by increasing respiratory chain activity or by ATP hydrolysis.

Mitochondria, conventionally considered the cell’s powerhouses, are intimately entwined

in apoptosis (Benardi et al., 2001). In particular, a mitochondria-dependent step,
involving outer-membrane permeabilization, appears related with most proapoptotic
stimuli. This process is controlled by the Bcl-2 family of proteins (Shi, 2001), as
activation members of the proapototic subgroup of this protein family, Bax a (Kroemer
and Reed, 2000) lead to the cytosolic release of mitochondrial intermembrane space
proteins. The mechanism by which these proteins induce the release of mitochondrial
proteins is still discussed. One proposed model involves rupture of the mitochondrial
outer membrane as a consequence of mitochondrial swelling after the opening of the
permeability transition (MMPT) pore (Marzo et al., 1998; Zamzani and Kroemer, 2001).
According to another model, proapoptotic Bcl-2 proteins like Bax induce a selective
process of outer membrane permeabiliztion independent of the PTP (Eskes et al., 1998)
through the formation of channels or pores (Martinou and Green, 2001), allowing the
selective release of proteins soluble in the intermembrane space; such as cytochrome c.

The involvement of mitochondria in apoptosis contribute to elucidate the mechanism by

which the release of apoptogenic factors can be modulated (Klohn et al ., 2003). The
experiments contribute to give an insight into the mechanism of mitochondrial membrane
permeabilization produced by apoptosis in vivo compared with opening of the MMPT
pore induced by Ca2+ in vitro.

This study was designed around the interest to see if chronic exposure to the antimalarial
drug, artesunate in the case of repeated use and/or overdose can induce/inhibit
mitochondrial membrane permeability transition, and to what extent; and then to check if
this can precipitate hepatotoxicity in the liver which is the major organ of detoxication.

In this study, the in vivo effect of various doses of artesunate (0.5mg, 1.0mg, 1.5mg,
2.0mg, 3.0mg and 5.0mg/kg body weight) on rat liver mitochondrial membrane
permeability transition (MMPT) were investigated. Membrane permeability transition
was estimated by the extent of mitochondrial swelling according to the procedure of
Lapidus and Sokolove (1993), who proposed that the protective effects of spermine on
the mitochondria reflect inhibition of the inner mitochondrial permeability transition. The
results revealed that the mitochondrial membrane of rats treated with artesunate at lower
doses of 0.5mg, 1.0mg and 1.5mg/kg body weight, in the absence of triggering agent
(-Ca2+) induced MMPT pore opening, but as the dose increased (2.0mg, 3.0mg and
5.0mg/kg body weight), there was a decrease in MMPT pore opening (inhibition of
MMPT pore in a dose-dependent manner). The presence of a triggering agent (+Ca2+) on
MMPT further induced pore opening in treated groups. In the untreated group, MMPT
remained intact except in the presence of a triggering agent (+Ca2+). The results from
liver (function) tests showed that glutamic-oxaloacetate transaminase (GOT), alkaline
phosphatase (ALP) and L-y-glutamyltransferase (L-y-GT) levels were significantly
higher (P<0.05) with the exception of glutamic-pyruvic transaminase (GPT) which was
lower in the plasma of artesunate-treated groups in comparison with the untreated,
healthy control group. Based on these results, there was no damage done to the liver by
2.0mg, 3.0mg and 5.0mg/kg daily doses of artesunate. Histological examination revealed
no visible lesion (NVL) in the livers from both treated and untreated rat livers. These
results therefore indicate that artesunate had inductive effect onMMPT which decreased
in a dose-dependent manner within the therapeutic range (2-5mg/kg body weight).