Multiple drug resistance (MDR) Tuberculosis (TB), leads to increased use of “second-line” drugs; one of the most effective is ethionamide (ETA). ETA is a prodrug metabolized by a mycobacterial flavin-containing monooxygenase (EtaA) as well human flavin-containing monooxygenases (FMOs). Of the five functional FMOs of humans, FMOs 1, 2, and 3 are the most important in metabolism of xenobiotics. FMO2 is a major isoform in the lung expressed in most mammals. In humans a polymorphism encoding inactive FMO2 is the common allele, while the wild type allele encoding active FMO2.1 has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. Human FMO2.1 is more efficient than EtaA in the first S-oxygenation of ETA to ETA S-oxide (ETASO). ETASO is a sulfenic acid and capable of redox-cycling with glutathione (GSH) producing oxidative stress and toxicity. We hypothesized that the FMO2 polymorphism will result in differences in the concentration and spectrum of ETA prodrug and metabolites. TB-infected individuals expressing active FMO2.1 will have reduced ETA efficacy in inhibition and killing of M. tuberculosis and enhanced oxidative stress, pulmonary toxicity and cell death in the host. To test this hypothesis, Fmo1/2/4 knockout mice modeling the FMO2*2 genotype were utilized to study ETA metabolism and toxicity compared to wild type mice in order to model humans expressing the FMO2*1 allele.
All mice were capable of metabolizing ETA to ETASO. Wild type mice had significantly higher plasma and epithelial lining fluid (ELF) levels of ETASO than ETA. In contrast, Fmo 1/2/4 knockout mice had higher plasma and ELF levels of ETA than ETASO. ETASO, a sulfenic acid, was significantly higher in wild type mice than knockout mice in both plasma and ELF. Investigation of the effects of higher levels of ETASO in wild type mice was warranted. Long-term ETA dosing was utilized to examine oxidative stress and toxicity. Compare to Fmo 1/2/4 knockout mice, wild type male and female mice showed more differentially expressed genes in lung related to oxidative stress and antioxidant defense pathways. Altered Fmo1, 2 and 4 expression was also found in lung and liver of long-term ETA-treated wild type mice in comparison to vehicle controls. To examine ETA therapeutic efficacy in the presence and absence of FMO, wild type and knockout mice were infected with M. avium and dosed with 50 mg/kg of ETA for 28-days and compared with vehicle-dosed mice for bactericidal effects. An ETA dose of 50 mg/kg did not significantly reduce the mycobacterium infection, leaving the fundamentally question of the role of FMO2 polymorphism on ETA efficacy unresolved. Though interestingly, significant higher bacterial load was observed in the ETA treated wildtype compared to ETA treated knockout mice which suggest that the presence of FMO in wildtype is making these mice more susceptible to bacterial infection when ETA is dosed.
Collectively these studies suggest that humans expressing active FMO2.1 may be at higher risk of ETA toxicity than individuals with inactive FMO2.2 that are undergoing long-term ETA treatment for MDR-TB. This work highlights the potential of the FMO2 human genetic on TB drug selection to maximize treatment efficacy and minimize toxicity. Another study of M. tuberculosis infected mice with 125 mg/kg dose of ETA is required to understand ETA therapeutic efficacy in the presence and absence of FMOs.