Investigation of in vivo unscheduled DNA synthesis in rabbit corneas following instillation of genotoxic agents.

PURPOSE: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents. MATERIALS AND METHODS: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography. RESULTS: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea. CONCLUSIONS: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

A Simple Ex Vivo Semiquantitative Fluorescent Imaging Utilizing Planar Laser Scanner: Detection of Reactive Oxygen Species Generation in Mouse Brain and Kidney.

OBJECTIVE: Oxidative stress plays an important role in the onset of many neuronal and peripheral disorders. We examined the feasibility of obtaining semiquantitative fluorescent images of reactive oxygen species (ROS) generation in mouse brain and kidney utilizing a planar laser scanner and dihydroethidium (DHE). METHODS: To investigate ROS generation in brain, sodium nitroprusside was injected into the striatum. Dihydroethidium was injected into the tail vein. After DHE injection, tissue slices were analyzed utilizing a planar laser scanner. For kidney study, cis-diamminedichloroplatinum [II] (cisplatin) was intraperitoneally administrated into mice. RESULTS: Clear and semiquantitative fluorescent images of ROS generation in the mouse brain and kidney were obtained. Furthermore, the fluorescence intensity was stable and not affected by fading. Sodium nitroprusside induced approximately 6 times the fluorescence accumulation in the brain. Cisplatin caused renal injury in all mice, and in comparison with control mice, more than 10 times fluorescence accumulation was observed in the renal medulla with tubular necrosis and vacuolization. CONCLUSIONS: We successfully obtained ex vivo semiquantitative fluorescent images of ROS generation utilizing a planar laser scanner and DHE. This simple method is useful for ROS detection in several ROS-related animal models and would be applicable to a variety of biochemical processes.

Understanding flow dynamics, viability and metastatic potency of cervical cancer (HeLa) cells through constricted microchannel.

To understand the burgeoning challenges of metastasis, a microchannel of 35 µm diameter, constricted to 7 µm for a distance of 200 µm in a total length of 3 mm, was designed and fabricated using a mask aligner made of polydimethylsiloxane (PDMS) to mimic in vivo capillaries. A thin glass cover-slide was mounted on top to monitor the motion of single or aggregated malignant HeLa cells (size 17-30 µm) microscopically through the constricted microchannel at a constant flow rate of 30 µl/h. Quantitative deconvolution of high-speed videographs of a single cell of 30 µm revealed cellular deformation while passing through constriction, having elongation index, average transit velocity and entry time of 2.67, 18 mm/s and 5.1 ms, respectively. Morphological analysis of live and apoptotic cells by dual staining with Acridine Orange/Ethidium Bromide demonstrated retention of a significant viable cell population after exit through the constriction and a viability index of 50% was quantified by dye exclusion assay. The cumulative data for microfluidic parameters, morphology and relevant metastatic MMP2 gene expression efficiency measured by real-time polymerase chain reaction revealed retention of virulence potency that could possibly cause metastasis, would be beneficial in developing futuristic MEMS device for cancer theranostics.

Chondrocyte Viability After a Simulated Blast Exposure.

OBJECTIVES: The effects of blast exposure have gained increasing interest in the military medical community with their continued occurrence on the battlefield. The impact of the direct and indirect energy imparted from blasts to hollow viscera, as well as closed head injuries, have been well studied. However, the injury to articular cartilage has not been investigated, despite previous correlations regarding the development of osteoarthritis. The purpose of this study was to assess the degree of injury to articular chondrocytes after exposure to a simulated blast overpressure wave. METHODS: Fresh juvenile porcine stifle joints were subjected to a simulated blast overpressure wave utilizing a custom fabricated blast simulator with compressed gases, within the reported range of observed battlefield blasts. Chondrocyte viability was assessed with live/dead staining using ethidium homodimer-2 and calcien acetoxymethylester stain and confocal laser scanning microscopy, calculated as a ratio of dead chondrocytes to live chondrocytes. Testing was performed at time points of 2, 4, and 8 hours after blast exposure and was compared with unblasted control samples. RESULTS: Chondrocyte viability decreased after exposure to a blast overpressure wave when compared with control samples. The amount of death was greater closer to the articular surface and dissipated with increasing tissue depth. Chondrocyte death increased with time after exposure. CONCLUSIONS: Chondrocyte death is present after exposure to a simulated blast wave. There is an inverse relationship between chondrocyte viability and the depth from the articular surface. Additional studies are needed to further characterize dose and time effects of blast exposure.

A novel and robust conditioning lesion induced by ethidium bromide.

Molecular and cellular mechanisms underlying the peripheral conditioning lesion remain unsolved. We show here that injection of a chemical demyelinating agent, ethidium bromide, into the sciatic nerve induces a similar set of regeneration-associated genes and promotes a 2.7-fold greater extent of sensory axon regeneration in the spinal cord than sciatic nerve crush. We found that more severe peripheral demyelination correlates with more severe functional and electrophysiological deficits, but more robust central regeneration. Ethidium bromide injection does not activate macrophages at the demyelinated sciatic nerve site, as observed after nerve crush, but briefly activates macrophages in the dorsal root ganglion. This study provides a new method for investigating the underlying mechanisms of the conditioning response and suggests that loss of the peripheral myelin may be a major signal to change the intrinsic growth state of adult sensory neurons and promote regeneration.

Dynamic optical imaging of metabolic and NADPH oxidase-derived superoxide in live mouse brain using fluorescence lifetime unmixing.

Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.

Synergistic effect of tetrandrine and ethidium bromide against methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) along with other resistant bacteria have become a significant social and clinical problem. Therefore, there is an urgent need to develop bioactive compounds from natural products as alternatives to the very few antibiotics that remain effective. Recently, the efflux mechanism has been identified as the main contributor to antibiotic resistance in bacteria. This study therefore aimed to evaluate tetrandrine (TET), an efflux pump inhibitor (EPI), as a potential antibiotic against MRSA. We investigated the antimicrobial activity of TET against 17 MRSA strains, of which 3 selected strains were studied in further detail using a time-kill assay. When these bacterial strains (1 × 10(6) colony-forming units (cfu)/ml) were incubated with TET in a time-kill assay, log-scale bactericidal activity was observed, which lasted for 24 hr. In addition, TET exhibits a synergistic effect when combined with the multi-drug resistance (MDR)-efflux pump substrate ethidium bromide (EtBr). Structure-function studies of the antibiotic activity of TET in combination with EtBr may lead to the discovery of more effective efflux pump inhibitors.

Effect of MOG sensitization on somatosensory evoked potential in Lewis rats.

Myelin oligodendrocyte glycoprotein (MOG) is commonly used as an immunogen to induce an immune response against endogenous myelin, thereby modeling multiple sclerosis in rodents. When MOG is combined with complete Freund's adjuvant (CFA), multifocal, multiphasic disease ensues; whereas when MOG is combined with incomplete Freund's adjuvant (IFA), clinical disease is usually absent. MOG-IFA immunized animals can be induced to have neurological disease after intraspinal injections of cytokines and ethidium bromide (EtBr). In this study, we investigated whether MOG-IFA immunized rats exhibited subclinical injury as defined by somatosensory evoked potential (SEP) recordings. The titration of anti-MOG-125 antibodies showed robust peripheral mounting of immune response against myelin in MOG-immunized rats. However the SEP measures showed no significant change over time. Upon injecting cytokine-EtBr in the spinal cord after MOG sensitization, the SEP recordings showed reduced amplitude and prolonged latency, suggestive of axonal injury and demyelination in the dorsal column, respectively. These findings were later confirmed using T2-weighted MRI and histological hematoxylin-eosin stain of the spinal cord. This report establishes that MOG-IFA immunization alone does not alter neuronal conduction in SEP-related neural-pathways and that longitudinal in-vivo SEP recordings provide a sensitive read-out for focal myelitis (MOG-IFA and intraspinal cytokine-EtBr) in rats.

High-dose morphine impairs vascular endothelial function by increased production of superoxide anions.

BACKGROUND: The effects of high-dose morphine on vascular endothelial function have not been previously shown. The authors hypothesized that the pro-oxidant effect of high-dose morphine impairs vascular endothelial function. METHODS: Mice were subjected to placebo or morphine (20 mg/kg intraperitoneal) injection for consecutive 14 days. Aortas were harvested for assessment of vasomotor function by isometric force recordings. Protein expression p47phox was determined by Western blotting. Generations of superoxide anions were detected under a confocal microscope. RESULTS: Compared with controls, contraction response to phenylephrine was significantly enhanced in the aorta of mice treated with high-dose morphine (maximal contractions were 150 +/- 26 vs. 261 +/- 32 mg, respectively; n = 5 or 6, P = 0.04). Endothelium-dependent relaxations to acetylcholine (10 to 10 m) were significantly reduced in morphine-treated animals but were normalized by superoxide scavenging. Fluorescent densities of dihydroethidium were increased in the aorta of morphine-treated mice. Aorta of mice treated with morphine expressed higher levels of p47phox (a major subunit of nicotinamide adenine dinucleotide phosphate oxidase). In cultured endothelial cells, morphine enhanced production of reactive oxygen species. CONCLUSIONS: Collectively, the authors' results showed that high-dose morphine impairs vascular endothelial function via attenuation of biologic activity of endothelium-derived nitric oxide. Chemical antagonism between superoxide anions generated by nicotinamide adenine dinucleotide phosphate oxidases may be the molecular mechanism responsible for the inactivation of endogenous nitric oxide after treatment with high-dose morphine.

Intravenous polyethylene glycol inhibits the loss of cerebral cells after brain injury.

We have tested the effectiveness of polyethylene glycol (PEG) to restore the integrity of neuronal membranes after mechanical damage secondary to severe traumatic brain injury (TBI) produced by a standardized head injury model in rats. We provide additional detail on the standardization of this model, particularly the use and storage of foam bedding that serves to both support the animal during the impact procedure-and as a dampener to the acceleration of the brass weight. Further, we employed a dye exclusion technique using ethidium bromide (EB; quantitative evaluation) and horseradish peroxidase (HRP; qualitative evaluation). Both have been successfully used previously to evaluate neural injury in the spinal cord since they enter cells when their plasma membranes are damaged. We quantified EB labeling (90 microM in 110 microL of sterile saline) after injection into the left lateral ventricle of the rat brain 2 h after injury. At six h after injection and 8 h after injury, the animals were sacrificed and the brains were analyzed. In the injured rat brain, EB entered cells lining and medial to the ventricles, particularly the axons of the corpus callosum. There was minimal EB labeling in uninjured control brains, limited to cells lining the luminal surfaces of the ventricles. Intravenous injections of PEG (1 cc of saline, 30% by volume, 2000 MW) immediately after severe TBI resulted in significantly decreased EB uptake compared with injured control animals. A similar result was achieved using the larger marker, HRP. PEG-treated brains closely resembled those of uninjured animals.

Expression of insulin-like growth factors in remyelination following ethidium bromide-induced demyelination in the mouse spinal cord.

Insulin-like growth factors, IGF-I and IGF-II, play important roles in development and myelination in the CNS, but little is known about the response of IGF after demyelination. The present study investigated the expression of IGF and their cognitive receptors in the process of remyelination following ethidium bromide (EBr)-induced demyelination in the adult mouse spinal cord. The present results, in a quantitative real-time PCR, showed significant increases in the levels of the mRNA for both IGF-I and IGF-II during both the demyelination and remyelination stages. The levels of IGF-I receptor (IGF-IR) mRNA increased from 10 days to 4 weeks after the EBr injection. The levels of IGF-II receptor (IGF-IIR) mRNA decreased for 6 days and then increased 10 days after the EBr injection. In situ hybridization studies showed the cells expressing IGF-I mRNA to be mainly macrophage-like cells, while those expressing IGF-II mRNA were predominantly Schwann cell-like cells invading the demyelinating lesion. The immunoreactivity for the IGF-IR and IGF-IIR increased in various kinds of cells within and around the demyelinating lesions from 6 days to 4 weeks after the EBr injection. These results suggest that locally produced IGF could partly be involved in some mechanisms underlying remyelination processes following the EBr-induced demyelination in the mouse spinal cord.

The reaction of glial progenitor cells in remyelination following ethidium bromide-induced demyelination in the mouse spinal cord.

The present study investigated how glial progenitor cells participated in the process of remyelination following ethidium bromide (EBr)-induced demyelination in the adult mouse spinal cord. In situ hybridization techniques for detecting mRNA for platelet-derived growth factor alpha receptor (PDGFalphaR) and proteolipid protein (PLP) were employed to identify glial progenitor cells and mature oligodendrocytes, respectively. During the demyelination stage and early stage of remyelination, large cells strongly expressing PDGFalphaR mRNA were observed in the border of the demyelinating lesion, and with immunohistochemistry they exhibited positive labeling of the astrocytic marker glial fibrillary acidic protein (GFAP). Other glial progenitor cells expressing PDGFalphaR mRNA proliferated around the lesion during the demyelination stage. During the remyelination stage, some PDGFalphaR mRNA-positive cells partly expressed mRNA for PLP in the periphery of the demyelinating lesion. These results suggest that PDGFalphaR mRNA-positive glial progenitor cells may give rise to both astrocytes and oligodendrocytes, which participate in remyelination following demyelination.

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle.

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

Sodium-potassium adenosine triphosphatase inhibition enhances membrane accumulation of DiI in rat hippocampus in vivo.

Transient brain ischemia induces significant alterations in lipid structures of neuronal membranes, which are believed to result from lipid peroxidation and free radical attack. Such a membrane structural change may serve as an important histological marker of cell injury. In the present study, we examined how the dynamics of DiI/membrane incorporation may reflect early membrane metabolism and dynamic changes following sodium-potassium pump inhibition. Ouabain (1mM) was stereotactically co-administered with either 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate DiI (50 microg/ml) or ethidium homodimer (4 microM) into the granule cell layer of the adult rat hippocampus. Tissue was cryosectioned and examined with epifluorescence microscopy at 1, 2, 3, 4, 6, 8 and 72h post-injection. Alternate sections were stained with thionine or haematoxylin and eosin to evaluate morphological changes. Ouabain-induced pump inhibition resulted in a dramatic increase in DiI fluorescence in granule cell layer neurons as early as 4h post-injection. This increase in DiI incorporation coincided both spatially and temporally with the appearance of reactive changes characterizing early neuronal injury. However, the fluorescence increase was not a result of membrane breakdown because ethidium homodimer, a membrane-impermeable nucleic acid probe used for labeling cells with compromised membranes, when applied in a similar fashion, failed to show any fluorescence changes. The results of this study suggest that pump inhibition results in a specific increase in membrane lipophilicity possibly due to altered lipid structure.

Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA).

Pharmacokinetic studies on the trypanocidal drug homidium bromide using a competitive enzyme immunoassay (detection limit 0.1 ng/mL) are reported for non-infected Friesian and Boran steers following treatment with homidium bromide at a dose of 1.0 mg/kg b.w. Following intravenous (i.v.) treatment of Friesian steers (n = 5), the mean serum drug concentrations were 31.9 +/- 2.1 and 3.9 +/- 0.4 ng/mL at 1 and 24 h, respectively. The decline in serum drug concentration was tri-exponential with half-lives of 0.064 +/- 0.037 h for t1/2 alpha, 7.17 +/- 1.87 h for t1/2 beta and 106.3 +/- 6.6 h for t1/2 gamma for distribution and elimination phases 1 and 2, respectively. Drug was detectable in serum for 17 days following treatment. The mean residence time (MRT) was 63.4 +/- 7.5 h. Following intramuscular (i.m.) treatment of Friesian steers (n = 5), the drug concentration at 1 h after treatment was 72.5 +/- 2.2 ng/mL. This declined to 9.8 +/- 1.8 ng/mL at 24 h. Low concentrations of between 0.1 and 0.3 ng/mL remained in circulation for up to 90 days post-treatment. Following intramuscular treatment of Boran steers (n = 5), the mean serum drug concentration at 1 h after treatment was 112.1 +/- 40.3 ng/mL. By 24 h after treatment, the concentration had fallen to 13.0 +/- 3.3 ng/mL. Thereafter, the serum drug concentration-versus-time profile and the pharmacokinetic parameters obtained following non-compartmental analysis were similar to those obtained following intramuscular treatment of Friesian steers.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle.

Two enzyme-linked immunosorbent assays (ELISA) for the determination of homidium in serum of treated cattle have been developed and evaluated. One is a direct competition (Assay 1) and the other an indirect competition assay (Assay 2). Both assays are highly sensitive with a limit of detection of 0.1 ng homidium per mL serum. Homidium levels were measurable in serum of cattle for over 2 months following administration of a single intramuscular (i.m.) dose at 1 mg/kg bodyweight. The level of sensitivity afforded by these assays makes them potentially useful tools in the pharmacokinetic evaluation of homidium and for investigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies.

Activation and proliferation of endogenous oligodendrocyte precursor cells during ethidium bromide-induced demyelination.

The adult brain contains a large population of glial cells with the properties of oligodendrocyte precursor cells (OPCs). The functions of this newly recognized class of glial cells in normal animals are unknown. Here, we analyzed the reactions of OPCs to a transient demyelination of the rat brainstem induced by the injection of ethidium bromide (EB) into the fourth ventricle. Within 22 h after EB injection, there is a 21% decrease in the number of OPCs within affected fiber tracts such as the spinal tract of the trigeminal nerve, most likely reflecting the toxic actions of EB. The surviving OPCs had enlarged cell bodies with fewer long processes and many membrane blebs. By 2 days after EB injection, these reactive OPCs had incorporated BrdU and increased in number. The increase in OPC cell number reached a maximum between 6-10 days after EB injection, at which time demyelination was complete. Myelin-specific marker antigens reappeared beginning at 12 days postinjection and the remyelination continued for up to 40 days. During remyelination, OPCs displayed a normal stellate morphology with an increased number of thin processes, many of which were closely associated with neurofilament-positive axonal profiles. The transient increase in the number of reactive OPCs within the demyelinated tissue and subsequent decrease in OPC number during remyelination demonstrates that the endogenous oligodendrocyte precursor population responds rapidly to the pathophysiological state of the brain. Demyelination generates a sufficient number of OPCs to participate in the repair of the demyelinated lesions.

Biodegradable polyesters for controlled release of trypanocidal drugs: in vitro and in vivo studies.

Copolymers of epsilon-caprolactone and L-lactide P(CL-LLA), epsilon-caprolactone and D,L-lactide P(CL-DLLA) and epsilon-caprolactone and trimethylene carbonate P(CL-TMC) were synthesized. The composition of comonomers and their sequence lengths were determined by means of 1H and 13C NMR measurements. The effect of the comonomer on the thermal properties was investigated by differential scanning calorimetry (DSC) analysis. The in vitro degradation of the rods obtained by melt extrusion of the synthesized copolymers and the commercial homopolymers poly(epsilon-caprolactone) P(CL) and poly(D,L-lactide) P(DLLA) was carried out in phosphate buffer (PB) pH 7.4 at 37 degrees C. The rate of degradation depends on comonomers and polymer composition. The in vitro release of the selected drugs, isometamidium chloride (IMM) and ethidium bromide (EtBr), from such devices was carried out under the same conditions as used for the in vitro degradation. The release experiments show that the release of IMM is faster than for EtBr. During the first stage, for IMM the release is governed by osmotic pressure whereas for EtBr the release is mainly diffusion-controlled. The in vitro release of these drugs is governed by polymer matrix degradation at the later stage of the release process. Comparative in vitro release study from the different polymers showed that the release depends mainly on the physical properties of the polymer. The in vivo experiments carried out in the field on cattle and in the laboratory on rabbits using the classical treatment (intramuscular injection) and the sustained release devices (SRD) subcutaneously implanted, showed that the prophylactic period is significantly enhanced in the case of SRD as compared to intramuscular injection. The comparative efficacy of SRD containing IMM and EtBr evaluated in the case of rabbits showed that, the SRD (IMM) prophylactic period is much longer than for SRD (EtBr).

Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia.

Ten trypanosome isolates were collected at random from cattle at Ghibe, Ethiopia, in February 1993 and all shown to be savannah-type Trypanosoma congolense. When inoculated into naïve Boran (Bos indicus) calves, all 10 isolates were resistant to diminazene aceturate (Berenil), isometamidium chloride (Samorin) and homidium chloride (Novidium) at doses of 7.0 mg/kg body weight (b.w.), 0.5 mg/kg b.w. and 1.0 mg/kg b.w., respectively. In order to determine whether this multiple-drug resistance was expressed by individual trypanosomes, clones were derived from two of the isolates and characterised in mice for their sensitivity to the three compounds; by comparison to drug-sensitive populations, the two clones expressed high levels of resistance to all 3 trypanocides. In experiments to characterise the uptake kinetics of [14C]-Samorin, the maximal rates of uptake (Vmax) for 4 Ghibe isolates ranged from 9.2 to 15.0 ng/10(8) trypanosomes/min. In contrast, Vmax for the isometamidium-sensitive clone T. congolense IL 1180 was 86.7 +/- 8.6 ng/10(8) trypanosomes/min. Lastly, molecular karyotypes were determined for eight isolates: seven different chromosome profiles were observed. These data indicate that in February 1993 there was a high prevalence of drug-resistant trypanosome populations with different chromosome profiles in cattle at Ghibe. Since a similar situation existed at the same site in July 1989, this suggests that the drug-resistance phenotype of trypanosomes at Ghibe had not altered over a 4 year period.