In 1990, Shapiro and Englund suggested that the main mode of action of ISMM was the cleavage of kDNA-topoisomerase complexes. This explanation was supported by Wells, Wilkes and Peregrine (1995) who showed that the trypanosome kinetoplast is the primary site of ISMM accumulation. The mechanism of resistance to ISMM, however, is less clear. Decreased levels of drug accumulation have been observed in drug-resistant populations of T. congolense (Sutherland et al., 1991) and later work found indirect evidence of an increased efflux of drug from resistant trypanosomes (Sutherland and Holmes, 1993). Recently, Mulugeta et al. (1997) showed that the maximal uptake rates (Vmax) of ISMM in resistant T. congolense were significantly lower than in sensitive populations. It remains to be shown whether this is caused by a decreased number of protein transporters of ISMM in the plasma membrane and/or by changes in the balance between influx and efflux. The role of nucleoside transporters in resistance to ISMM by T. congolense remains to be examined, although changes in these transporters have been associated with resistance to arsenical drugs in T. brucei (Carter and Fairlamb, 1993; Carter, Berger and Fairlamb, 1995; Ross and Barns, 1996). More recently, changes in mitochondrial electrical potential have been demonstrated in ISMM-resistant T. congolense by Wilkes et al. (1997).
Although contradictory observations have been reported on the genetic stability of ISMM resistance, recent field observations in Ethiopia, based on cloned populations, showed that the drug-resistant phenotype of T. congolense had not altered over a period of four years (Mulugeta et al., 1997).
Although their mutagenic activity has been known for a long time (MacGregor and Johnson, 1977), homidium chloride and especially homidium bromide or ethidium are still widely used as trypanocidal drugs. The mechanism of their antitrypanosomal action is not well understood. However, it has been shown that the drugs interfere with glycosomal functions, the function of an unusual adenosine monophosphate-(AMP) binding protein, trypanothione metabolism and the replication of kinetoplast minicircles (Wang, 1995). The mechanism of resistance by trypanosomes to these drugs is unknown. There are indications, however, that it is similar to that described for ISMM (Peregrine, Gray and Moloo, 1997).
Although diminazene probably exerts its action at the level of the kinetoplast DNA, this has not been proven in vivo, and other mechanisms of action cannot be excluded (Peregrine and Mamman, 1993). Similarly the molecular basis of resistance to diminazene in trypanosomes is not clear. Carter, Berger and Fairlamb (1995) showed that the accumulation of diminazene was markedly reduced in arsenical-resistant T. brucei brucei owing to alterations in the nucleoside transporter system (P2). However, there might be other resistance mechanisms (Zhang, Giroud and Baltz, 1992).
Similarly to ISMM, contradictory reports have also been published on the stability of resistance to diminazene. Mulugeta et al. (1997), however, showed that the phenotype of multiple drug-resistant (including diminazene) T. congolense remained stable over a period of four years.
In conclusion, it is clear that much more work is required in order to elucidate the mechanism of resistance to the three currently available trypanocidal drugs. Such studies, as well as being of great value in their own right, may also provide novel methods for the detection of drug-resistant trypanosomes in the future.
The same is true for the genetics of drug resistance in trypanosomes. Hayes and Wolf (1990) distinguish three major types of genetic change that are responsible for acquired drug resistance: mutations or amplifications of specific genes directly involved in a protective pathway; mutations in genes that regulate stress-response processes and lead to altered expression of large numbers of proteins; and gene transfer. Gene amplification under conditions of drug pressure is well known in Leishmania spp. and has also been demonstrated in trypanosomes, but until now there is no evidence that this occurs in the latter parasites as a mechanism of drug resistance (Ross and Sutherland, 1997). The current possibilities to insert or delete genes will certainly lead to a better insight into the resistance mechanisms (Ten Asbroek, Ouellette and Borst, 1990; Gaud et al., 1997). Other aspects, such as the stability of drug resistance, its mono- or polygenic nature, dominance or recessiveness, also need to be examined, because of their far-reaching impact on the control of resistance.
