Rhus chinensis Mill. fruits alleviate liver injury induced by isoniazid and rifampicin through regulating oxidative stress, apoptosis, and bile acid transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhus chinensis Mill. is a species of the genus Rhus belonging to the family Anacardiaceae. Its fruits used to treat/prevent liver related diseases (e.g., jaundice and hepatitis) in folk medicine. Otherwise, the effects and underlying mechanisms of the fruits on the prevention of isoniazid and rifampicin-caused liver injury have not been investigated. AIM OF THE STUDY: To study the preventive effects and mechanisms of the Rhus chinensis Mill. fruits on isoniazid and rifampicin-caused liver injury. MATERIALS AND METHODS: This experiment was based on rifampicin (75 mg/kg/day) and isoniazid (75 mg/kg/day)-induced liver damage model to explain the pharmacological effects of Rhus chinensis Mill. fruits. The prevention of the extract from Rhus chinensis Mill. fruits on isoniazid and rifampicin-caused liver injury were evaluated using biochemical parameters, histopathological analysis, and immunofluorescence technique. Apart from that, the potential molecular mechanisms were elucidated by analyzing the expression of such crucial proteins participated in oxidative stress, apoptosis, and bile acid transport. RESULTS: The extract from Rhus chinensis Mill. fruits significantly reduced the levels of ALT, AST, TBIL, ALP and MDA. Besides, the extract, especially 800 mg/kg b.w., was remarkably decreased the content of TNF-α,IL-6 and IL-1ß, restored the levels of GSH and SOD. The results of Western blot also presented that the extract could activate the Nrf2 protein pathway and inhibit the expression of CYP2E1 to reduce oxidative stress. Meanwhile, the extract significantly up-regulated the expressions of BSEP and Mrp2 to regulate the transport of bile acid, and alleviated the cellular apoptosis via adjusting the expression of Bax and Bcl-2 proteins. CONCLUSIONS: Rhus chinensis Mill. fruits can prevent the liver injury induced by isoniazid and rifampicin in mice through adjusting the expressions of multiple proteins in oxidative stress, apoptosis, and bile acid transport pathways. This paper may provide scientific basis for the fruits as a Chinese medicine to prevent/cure liver injury.

Ameliorative potential of Adhatoda vasica against anti-tubercular drugs induced hepatic impairments in female Wistar rats in relation to oxidative stress and xeno-metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Adhatoda vasica Nees is widely used herb of indigenous system to treat various ailments especially upper respiratory tract infections. Not only, anti-tubercular efficacy of crude extract and phytoconstituents of A. vasica has been documented but its hepatoprotective role against various drugs mediated hepatic alterations in different animal models has also been observed. BACKGROUND AND PURPOSE: Isoniazid, rifampicin and pyrazinamide (H-R-Z) are anti-tubercular drugs normally prescribed by health professionals for the treatment of tuberculosis, however along with their medical effectiveness these drugs also exhibit hepatotoxicity among TB patients. Unexpectedly, substantial toxicological data on the metabolism of anti-TB drugs are available but the mystery behind these xenobiotics is too complex and partly implicit. In this study, we further explored the hepatotoxic effects of these xeno-metabolic products and their amelioration by Adhatoda vasica Nees by elucidating its mechanistic action. METHODS: We generated a hepatotoxic rodent model by oral administration of H, R and Z (30.85, 61.7 and 132.65 mg/kg body weight) drugs for 25 days in Wistar rats. Additionally, to achieve hepatoprotection two different doses of Adhatoda vasica Nees ethanolic leaf extract (200 and 300 mg/kg body weight) were used along with H-R-Z dosage, orally and once daily for 25 days and tried to ascertain their mechanistic action. For this, initially phytoconstituents of the extract were evaluated followed by extract standardization using RP-HPLC and FTIR methods. Furthermore, antioxidant activity of the extract was analyzed by DPPH assay. Finally, different treated groups were analyzed for hepatic oxidative stress markers, antioxidant markers, histopathological changes and gene expression study including CYP2E1, CYP7A1, NAT, NR1I2 and UGT1A1 genes involved in phase I and phase II xeno-metabolism. RESULTS: Estimated content of vasicine in RP-HPLC method and free-radical scavenging activity in DPPH assay was found to be 134.519 ± 0.00269µg/10mg of leaf extract and 47.81 µg/mL respectively. In H-R-Z treated group, a significant increase in the levels of thiobarbituric acid, significant reduction in the levels of GSH, and enzymatic markers and marked changes in hepatic histological architecture were observed. In addition, there was significance up-regulation of CYP7A and NAT genes, down-regulation of CYP2E1 gene and insignificant expression levels of NR1I2 and UGT1A1 genes were observed in H-R-Z group. Conversely, high dose of A. vasica extract effectively diminished these alterations by declining oxidative stress and boosting of antioxidant levels. In addition, it acted as bi-functional inducer of both phase I (CYP2E1) and phase II (NAT and UGT1A1) enzyme systems. CONCLUSION: Hence, we concluded that anti-TB drugs exposure has potential to generate reactive metabolites that eventually cause hepatotoxicity by altering oxidant-antioxidant levels and their own metabolism. This study not only emphasized on xeno-metabolism mediated hepatic alterations but also explore the benefit of A. vasica on these toxic insults.

Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn(III): First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location.

Isoniazid (INH), the most-widely used anti-tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon-centered isonicotinic acyl radical, which was considered to be responsible for its anti-tuberculosis activity. However, it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal- or proximal-nitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn(III) pyrophosphate. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.

Theoretical insights on the binding of isoniazid to the active site residues of Mycobacterium tuberculosis catalase-peroxidase.

Isoniazid (INH) is known to cause the exclusive lethal action to Mycobacterium tuberculosis (M. tb.) cells because of the pathogen's own catalase-peroxidase (katG) enzyme that converts INH to a very reactive radical. Thus, in order to gain insights on the interaction of INH with the individual active site residues (Res) of katG, this study presents a computational approach via molecular docking and density functional theory (DFT) using augmented models to study the individual INH-Res interactions. Seven amino acid residues directly interacts with INH: Arg104, Asp137, His108, Ile228, Trp107, Tyr229, and Val230. The residues with the highest interaction energies are Arg104 (-39.64 kcal/mol) and Asp137 (-32.85 kcal/mol) mainly due to strong ion-dipole and H-bonding interactions present in the complexes, while the weakest interaction was observed for Ile228 (-13.78 kcal/mol). Molecular electrostatic potential surface revealed complementary regions for dipole interactions and charge distribution analysis further shows that INH generally donates electrons to the residues. The results in this study agrees with the previous experimental findings and provides new insights into the catalase dependent activation of INH and the methods presented may be valuable in the study of biological metabolism of molecules.

A High Dose of Isoniazid Disturbs Endobiotic Homeostasis in Mouse Liver.

Overdose of isoniazid (INH), an antituberculosis drug, can be life-threatening because of neurotoxicity. In clinical practice for management of INH overdose and acute toxicity, the potential of INH-induced hepatotoxicity is also considered. However, the biochemical basis of acute INH toxicity in the liver remains elusive. In the current study, we used an untargeted metabolomic approach to explore the acute effects of INH on endobiotic homeostasis in mouse liver. We found that overdose of INH resulted in accumulation of oleoyl-l-carnitine and linoleoyl-l-carnitine in the liver, indicating mitochondrial dysfunction. We also revealed the interactions between INH and fatty acyl-CoAs by identifying INH-fatty acid amides. In addition, we found that overdose of INH led to the accumulation of heme and oxidized NAD in the liver. We also identified an INH and NAD adduct in the liver. In this adduct, the nicotinamide moiety in NAD was replaced by INH. Furthermore, we illustrated that overdose of INH depleted vitamin B6 in the liver and blocked vitamin B6-dependent cystathionine degradation. These data suggest that INH interacts with multiple biochemical pathways in the liver during acute poisoning caused by INH overdose.

On-Chip Construction of Liver Lobule-like Microtissue and Its Application for Adverse Drug Reaction Assay.

Engineering the liver in vitro is promising to provide functional replacement for patients with liver failure, or tissue models for drug metabolism and toxicity analysis. In this study, we describe a microfluidics-based biomimetic approach for the fabrication of an in vitro 3D liver lobule-like microtissue composed of a radially patterned hepatic cord-like network and an intrinsic hepatic sinusoid-like network. The hepatic enzyme assay showed that the 3D biomimetic microtissue maintained high basal CYP-1A1/2 and UGT activities, responded dynamically to enzyme induction/inhibition, and preserved great hepatic capacity of drug metabolism. Using the established biomimetic microtissue, the potential adverse drug reactions that induced liver injury were successfully analyzed via drug-drug interactions of clinical pharmaceuticals. The results showed that predosed pharmaceuticals which agitated CYP-1A1/2 and/or UGT activities would alter the toxic effect of the subsequently administrated drug. All the results validated the utility of the established biomimetic microtissue in toxicological studies in vitro. Also, we anticipate the microfluidics-based bioengineering strategy would benefit liver tissue engineering and liver physiology/pathophysiology studies, as well as in vitro assessment of drug-induced hepatotoxicity.

Attenuation of anti-tuberculosis therapy induced hepatotoxicity by Spirulina fusiformis, a candidate food supplement.

Therapy using Isoniazid (INH) and Rifampicin (RIF) leads to induction of hepatotoxicity in some individuals undergoing anti-tuberculosis treatment. In this study, we assessed the effect of Spirulina fusiformis on INH and RIF induced hepatotoxicity in rats compared with hepatoprotective drug Silymarin. Induction of hepatotoxicity was measured by changes in the liver marker enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase). The antioxidant status was also analyzed in liver tissue homogenate and plasma by measurement of superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, and lipid peroxidation levels. We also aimed to study the binding and interactions of the transcription factors Pregnane X Receptor (PXR) and Farnesoid X Receptor (FXR) with INH, RIF, and representative active compounds of Spirulina fusiformis by in silico methods. The administration of INH and RIF resulted in significant (p < 0.05) decrease in the antioxidant levels and total protein levels. There was also a significant (p < 0.05) increase in the levels of liver marker enzymes. Spirulina fusiformis was seen to protect the parameters from significant changes upon challenge with INH and RIF in a dose-dependent manner. This was corroborated by histological examination of the liver. The results of the in silico analyses further support the wet lab results.

Assessment of the antimutagenic effect of Doash tea extract fractions.

Origanum majorana L (Doash) is one of the traditional remedy that is used as a tea and to treat ailments, in the Kingdom of Saudi Arabia. The present study has attempted to evaluate the inhibitory action of Doash fractions on the bioactivation of selected food mutagens and direct-acting mutagens. Four Sallmonella bacterial strains (TA98, TA97, TA100 and TA1530) were used in the present study. These strains contain different mutations in the histidine operon, allowing the bacteria to detect different types of mutation. The two strains (TA98 and TA97) are capable of detecting frameshift mutations, while TA100 and TA1530 are able to detect base-pair substitutions. The liver homogenate and other subcellular fractions were prepared. Identification of Doash fractions was conducted using the high-performance liquid chromatography/mass spectrometry system. The results of the present study demonstrated that the Doash tea fraction components have the ability to reduce the in vitro mutagencity of several promutagens, which were employed in this study. Fraction No. 5 (with the highest content of solid) was the most potent inhibitor of the mutagenicity of all promutagens employed in this study. The antimutagenic effect of Doash tea extract, and its various fractions, was pronounced, indicating that the metabolism of cytochrome P450 1A2 isozyme is likely to be inhibited.

In vitro evaluation of inhalable isoniazid-loaded surfactant liposomes as an adjunct therapy in pulmonary tuberculosis.

In this study, exogenous pulmonary surfactant was evaluated as an inhalable drug carrier for antitubercular drug isoniazid (INH). Isoniazid-entrapped liposomes of dipalmitoylphosphatidylcholine (DPPC) (the most abundant lipid of lung surfactant and exogenous surfactant) were developed and evaluated for size, drug entrapment, release, in vitro alveolar deposition, biocompatibility, antimycobacterial activity, and pulmonary surfactant action. Isoniazid-entrapped DPPC liposomes were about 750 nm in diameter and had entrapment efficiency of 36.7% +/- 1.8%. Sustained release of INH from DPPC liposomes was observed over 24 h. In vitro alveolar deposition efficiency using the twin impinger exhibited approximately 25-27% INH deposition in the alveolar chamber upon one minute nebulization using a jet nebulizer. At 37 degrees C, the formulation had better pulmonary surfactant function with quicker reduction of surface tension on adsorption (36.7 +/- 0.4 mN/m) than DPPC liposomes (44.7 +/- 0.6 mN/m) and 87% airway patency was exhibited by the formulation in a capillary surfactometer. The formulation was biocompatible and had antimycobacterial activity. The isoniazid-entrapped DPPC liposomes could fulfill the dual purpose of pulmonary drug delivery and alveolar stabilization due to antiatelectatic effect of the surfactant action which can improve the reach of antitubercular drug INH to the alveoli.

[Dissolved ozone treatment-induced change in the resistance of multi-resistant mycobacterial strain to isoniazid and rifampicin]. / Izmenenie ustoichivosti k izoniazidu i rifampitsinu poliresistentnogo shtamma mikobakterii tuberkuleza posle obrabotki rastvorennym ozonom.

For 60 minutes, a mycobacterial (MBT) clinical strain resistant to streptomycin (S), rifampicin (R), isoniazid (I) was treated with dissolved ozone (PO3) at the concentration used for intravenous injection in the clinic. Then the strain was added to the Löwenstein-Jesen solid medium containing different concentrations of antituberculous agents. Following 3 weeks, drug sensitivity was determined by the number of grown colonies. Then MBT were retreated with PO3 in the same fashion, by repeating the cycle three times. At week 3, a growth of over 100 colonies was recorded in all control cultures. After each PO3 treatment of the strain, there was a reduction in its resistance to I and R. After triple treatment, MBT sensitivity to I completely recovered. In the R-containing media, there was also decrease in drug resistance, but the latter remained high (640 mu/ml). S resistance substantially lowered after the second PO3 treatment, but it restored after the third one. A mechanism responsible for lower MBT resistance to I and R under the action of "therapeutical" concentrations of PO3 is analyzed. The paper discusses whether MBT resistance can be changes at the phenotypic level rather that at the genetic one.

Iron chelators of the pyridoxal isonicotinoyl hydrazone class. III. Formation constants with calcium(II), magnesium(II) and zinc(II).

Formation constants for the calcium(II), magnesium(II) and zinc(II) complexes of the orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH) and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone (PpMBH) and pyridoxal m-fluorobenzoyl hydrazone (PmFBH) have been determined by potentiometry at 25 degrees C and I = 0.1 M [KNO3]. The four ligands bind calcium(II) weakly and magnesium(II) only slightly more strongly, as a 1:1 complex which is formed at pH greater than 8. The chelation of zinc(II) for all the ligands studied was greater than that for calcium(II) and magnesium(II), with complexation generally becoming significant at about pH 5. Thus, chelation of zinc(II) but not calcium(II) or magnesium(II) at physiological pH, 7.4 may be expected. Calculated values of the concentration of uncomplexed metal ion indicate that the selectivity of these ligands towards Fe(III) is comparable to that of the clinically used chelator desferrioxamine.

Mutagenic activity of isoniazid in the mouse.

Isoniazid was tested for genetic activity in the mammalian spot test by treating in utero mouse embryos, derived from the crosses C57 B1/6J X T-stock and NMRI X DBA/2, heterozygous for various loci affecting coat color. Induction of genetic changes is seen by expression of recessive alleles in colored spots within the fur of the adult animal in a non-agouti background. Injecting mothers with isoniazid (100 mg/kg ip), an expression of recessive loci was found in 7.41% of the F1 animals in the cross C57 B1/6J X T-stock, while in the NMRI X DBA/2 cross 2.92% animals with recessive spots were detected. The control frequencies were respectively 0 and 0.78% animals with recessive spots. The positive control substance, ethyl methanesulfonate (100 mg/kg ip), yielded 5.97% and 4.71% animals carrying a recessive spot in the fur, respectively. These studies clearly demonstrate for the first time the induction of genetic damage other than cytological damage in somatic cells of mice by isoniazid.

Inactivation of isoniazid by condensation in a syrup preparation.

This paper reports the gross and rapid condensation of isoniazid in a commercial black-currant-flavoured syrup. In vitro studies showed that the condensation was due, at least partly, to the glucose contained in the syrup, paper chromatography having demonstrated the presence of D(+)-glucose isonicotinoyl hydrazone. Controlled studies in human beings showed that the absorption of isoniazid from the preparation was considerably impaired by this condensation.It is concluded that sugars such as glucose, fructose, and sucrose-especially glucose-should not be used in isoniazid syrup preparations, and it is suggested that sorbitol, a stable non-carbonyl compound, might be a suitable substitute.