Browsing by Author "Motha, Khetiwe"

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    A zebrafish larval model for drug-induced hepatic Injury due to first-line antituberculosis drugs and possible prevention/treatment with N-acetyl-cysteine
    (Stellenbosch : Stellenbosch University, 2022-11) Motha, Khetiwe; Kellermann, Tracy; Smith, Carine; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Background Tuberculosis (TB) is ranked as the second most deadly infectious disease worldwide. First-line TB medication is associated with the development of drug-induced liver injury (DILI). The hepatotoxicity from this combination therapy is mostly due to pyrazinamide (PZA), isoniazid (INH) and rifampicin (RIF), however, no information has been reported on the hepatotoxicity potential of ethambutol (EMB). DILI typically occurs within the initial few weeks of the intensive phase of therapy. The only way to treat DILI is by stopping the medication and considering a liver transplant in the case of liver failure. Halting treatment can lead to the development of multi-drug resistant TB (MDR-TB). Therefore, close monitoring during therapy is required. Moreover, a preventative intervention needs to be put in place in order to prevent TB-DILI from developing in the first place. Acetaminophen (APAP) has been shown to cause DILI due to the accumulation of the drug’s toxic metabolites in the liver following overdose of the drug. N- acetylcysteine (NAC) is an effective treatment and works by replenishing cellular glutathione in hepatocytes, thereby preventing liver injury from progressing to liver failure. It is therefore hypothesized that NAC may be able to reverse liver injury due to first line TB medication since it has been shown to be highly effective in the treatment of DILI associated with APAP, and has in fact become the standard of care. Previous studies have reported the potential of NAC in treating TB-dug associated DILI. However, this needs to be confirmed through further studies. Therefore, new models are needed for predicting which therapeutic compounds could cause DILI in humans, and new markers and mediators of DILI need to be identified. The physiological and metabolic processes in zebrafish (Danio rerio) are similar to that of humans and the transparency of the zebrafish larvae makes this vertebrate a suitable model for studying TB-DILI mechanism and treatment. This study therefore aims to develop a zebrafish larval model (<5dpf) for DILI due to TB drugs, using APAP as a positive control that is known to cause DILI and to investigate the potential of NAC in preventing liver injury. Methods High-performance liquid chromatography (HPLC) analysis of INH, PZA and RIF was performed using previously developed methods, while liquid chromatography tandem mass spectrometry (LC-MS/MS) using a previously developed method was used for the evaluation of EMB. An HPLC method for the quantitation of APAP was developed and partially validated. The method used a Shimadzu HPLC system coupled with a PDA detector. Successful separation was achieved by isocratic elution on a reverse-phase Venusil XBP C18 (4.6 X 100 mm, 5 μm) column using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (82:18; A:B, v:v) at 0.650 ml/min flow rate, detection wavelength of 247 nm, column oven temperature of 28°C and injection volume of 10 μl. The chromatographic retention time was consistent at 3.26 min. The calibration curve covered a range of 3.13 to 200 μg/ml with a quadratic regression weighted 1/c (c: concentration). These analytical methods were used to determine the solubility and stability of these drugs in E3 medium at 28°C for 3 days. Thereafter, dose response experiments were performed for all drugs to obtain the dose (s) that resulted in liver steatosis as an indicator of DILI using Oil red O positive liver stains and EthoVision movement tracking software (for NAC) as endpoints. Optimisation of the doses and the evaluation of the ability of NAC to prevent or reverse TB drug-associated DILI was attempted in zebrafish larvae. For NAC, APAP, INH and PZA, previously reported zebrafish larval doses for each drug were used a as reference for the study. However, for rifampicin (RIF) and EMB, the doses were adjusted according to the human dose. Results Solubility and stability data showed that APAP, INH, PZA and EMB were soluble in E3 embryo water and stable at 28˚C for 3 days. However, RIF remained insoluble and unstable in E3 medium at 28°C after 24 hours, even with the addition of ascorbic acid at 20 μg/ml. Doses of 1 mM, 7 mM, 10 μM, 1.2 mM and 0.5 mM were selected as the doses associated with DILI for INH, PZA, RIF, EMB and APAP, respectively. EthoVision data showed that 12 μM and 16 μM NAC resulted in irritation and toxicity, respectively. However, 8 μM NAC was shown to be consistent with the negative control and was therefore selected as the safe dose for NAC. Due to time constraints and difficulties in breeding, NAC was not evaluated for its ability to prevent or reverse DILI due to first-line TB drugs and this will be elucidated in future. Conclusion A time efficient, economical zebrafish larval model for DILI was successfully developed. Current data illustrates that this model is able to accurately simulate known toxicity effects of first-line antituberculosis drugs on the liver. Furthermore, this model can be used to evaluate potential treatment interventions and prophylaxis for the reversal and/or prevention of DILI.