Betty-Ann Hoener

Changes in plasma protein binding have little clinical relevance

Clinical Pharmacology & Therapeutics (2002) 71, 115–121; doi: 10.1067/mcp.2002.121829

Nitrofurantoin Produces Oxidative Stress and Loss of Glutathione and Protein Thiols in the Isolated Perfused Rat Liver

The effects of 150, 600 or 1,200 nmol/ml of nitrofurantoin on glutathione (GSH), glutathione disulfide (GSSG), protein thiols (PSH) and cell integrity were studied in the isolated perfused rat liver. Nitrofurantoin produced a dose-dependent, up to 3-fold, increase in bile flow and a marked, up to 150-fold, increase in biliary excretion of GSSG. By the conclusion of the experiment, tissue levels of GSH had fallen to 81 +/- 14, 41 +/- 10 and 16 +/- 5% of control values at the three dose levels. Tissue levels of GSSG rose from 18.3 +/- 2.3 to 45.3 +/- 8.0 nmol/g and from 20.0 +/- 6.0 to 187 +/- 47 nmol/g within 15 min at the two higher doses, but fell to initial levels by the end of the experiment. Only at the 1,200-nmol/ml dose did the tissue levels of PSH decline, to 64 +/- 14% of initial values, by the end of the experiment. Lactate dehydrogenase and transaminases were found in the perfusate only after the GSH and PSH levels had fallen. After a 60-min exposure to 1,200 nmol/ml of NFT followed by blank perfusate for 3 h, massive engorgement of the liver was noted. Microscopic examination revealed extensive interstitial edema, nuclear pyknosis, cytoplasmic shrinkage and vacuolization, and mitochondrial dense deposits. We conclude that toxic doses of nitrofurantoin can produce cellular depletion of GSH and PSH which, if not the direct cause, at least signal the loss of cell viability.

Transport and metabolism of cyclosporine in isolated rat hepatocytes: The effects of lipids

The effects of lipids on the uptake and metabolism of cyclosporine (CyA) were investigated in isolated rat hepatocytes. In the absence of lipids, CyA was rapidly taken up (reaching apparent steady state within 5 min) and highly associated with the cells (more than 80%). The CyA uptake was concentration independent over the concentration range studied (0.6 to 11.2 micrograms/mL). Metabolism, however, was relatively slow and saturable. Except for cholesterol (at concentrations up to 15.5 mM), all lipids tested [oleic acid; low density lipoproteins (LDL); and high density lipoproteins (HDL)] reduced CyA cell uptake as well as its metabolism in a concentration-dependent manner. The effects of LDL were much more pronounced when compared to those of HDL and oleic acid. At an LDL concentration of 1 microM, drug uptake, indicated by the cell-associated concentration at steady state, was about 49% of the control value, while CyA metabolism was inhibited completely. Drug uptake of about 82 and 91% and CyA disappearance of 75 and 68% of the relevant control values were observed with HDL and oleic acid at concentrations of 10 microM and 0.7 mM, respectively. Apparently, lipids decreased CyA metabolism by reducing the concentration of CyA available for transport into the cells. These findings further support the suggestion of an important role for plasma lipids in the disposition of CyA.

Evidence for the Involvement of a Nitrenium Ion in the Covalent Binding of Nitrofurazone to DNA

We have shown that the xanthine oxidase-catalyzed anaerobic reduction of nitrofurazone in the presence of added DNA leads to the formation of covalently bound adducts. Further, by systematically decreasing the pH of the reaction mixture, we have demonstrated that generation of the reactive species is facilitated under mildly acidic conditions. From these observations, we conclude that it is the nitrenium ion formed from nitrofurazone which binds to DNA.

Nitrofurazone: Kinetics and oxidative stress in the singlepass isolated perfused rat liver

The disposition of the antibiotic nitrofurazone was studied in the singlepass isolated perfused rat liver. Both the effects of the steady-state level of drug and the composition of the perfusate were evaluated. The higher level (120 micrograms/ml) of nitrofurazone in a perfusion medium lacking the glutathione (GSH) precursors, glycine, glutamic acid and cysteine, caused a marked increase in bile flow (from 1.01 +/- 0.07 to 2.33 +/- 1.07 microliters/min/g), massive biliary efflux of glutathione disulfide (GSSG) (from 0.55 +/- 0.07 to 60.6 +/- 25.4 nmol/min/g) and a sharp decline in the caval efflux of GSH (to undetectable levels) and the tissue level of GSH (from 5.74 +/- 0.20 to 2.68 +/- 0.13 mumol/g). Even after the drug was discontinued, these parameters were not restored to control levels. The lower level (30 micrograms/ml) of nitrofurazone with or without amino acid supplementation and the higher level with supplementation induced less dramatic effects. Using [35S]methionine, a new conjugated metabolite of nitrofurazone and glutathione was detected. The data suggest that the toxicity of the reactive oxygen species generated by the redox cycling of the nitro group and the reactive metabolites generated by further reduction of nitrofurazone can be mitigated by adequate glutathione levels, but that livers lacking sufficient glutathione to scavenge these reactive species may be damaged.

Nitrofurazone disposition by perfused rat liver. Effect of dose size and glutathione depletion.

The disposition of nitrofurazone was studied in the isolated perfused rat liver using a recirculating system. The drug was administered as a bolus in two different doses (3.5 and 14 mg: initial concentrations 0.35 and 1.4 mM respectively), and its disappearance was monitored by analyzing perfusate samples at various times. Biliary excretion and bile flow were also measured. In all experiments perfusate disappearance was monoexponential, and no significant difference was found between the two doses (T 1/2: 5.34 +/- 2.03 and 6.19 +/- 1.47 min for 14 and 3.5 mg respectively). Bile flow increased more than 2-fold 5-10 min after administration of the drug and subsequently returned to control levels. The increase in bile flow was dose-related and paralleled the excretion of the parent drug in the bile; however, of the total dose administered, only 0.27 +/- 0.04% was excreted unchanged in bile, thus ruling out an osmotic choleresis due to the parent drug. Since nitrofurazone may be excreted in part as a glutathione conjugate, this or other metabolites could have caused an osmotic choleresis. This hypothesis was tested by administering diethylmaleate which causes glutathione depletion. Although the initial bile flow in treated livers was not different from untreated livers, bile flow did not increase after administration of nitrofurazone. In addition, the perfusate half-life of nitrofurazone was increased (18.18 +/- 1.30 min, P less than 0.005). These results suggest that nitrofurazone is cleared rapidly by the liver and that glutathione plays an important role in its disposition.

An isolated perfused lung model with real time data collection and analysis of lung function.

Our primary purpose in making this report has been to describe an isolated perfused lung system which permits the real time collection and analysis of lung mechanical functioning. The distinct advantage of our system lies in its capacity for breath by breath data acquisition and analysis. In addition, because of the modular nature of the components, the system can be readily expanded or contracted depending on the type of experiment being conducted. As configured, the lung mechanic parameters of air flow, lung volume, transpulmonary pressure, pulmonary artery pressure, weight, resistance, elastance (inverse of compliance), and positive end expiratory pressure were monitored, recorded, and evaluated simultaneously throughout the experimental period. We present the results of a 3-h study with control lungs illustrating the stability of these measurements throughout the entire period. Also included is a brief discussion of 3-h studies which show a progressive loss of viability in lungs treated with the redox cycler nitrofurantoin.

Oxidative Metabolites of 5-Nitrofurans

We synthesized the 4-hydroxy derivatives of nitrofurazone, furazolidone and nitrofurantoin. Then we dosed rats orally with these antibiotics and isolated the intensely yellow, polar metabolites from their urine. A comparison of the ultraviolet and nuclear magnetic resonance spectra of these metabolites with the corresponding synthetic derivatives confirmed that the metabolites are 4-hydroxynitrofurazone, 4-hydroxyfurazolidone and 4-hydroxynitrofurantoin.

α-Tocopherol succinate does not mitigate nitrofurantoin-induced changes in the glutathione and protein thiol status of the isolated perfused rat liver

Because it had been suggested that α-tocopherol might protect protein thiols (PSH) from oxidation, we were interested in determining if α-tocopherol could prevent or mitigate nitrofurantoin-induced changes in the glutathione (GSH) or PSH status of the liver. Isolated rat livers were perfused in the single pass mode for 310 min with Krebs-Henseleit buffer or the same buffer containing 25 nmol/ml of α-tocopherol succinate. Treated livers were exposed to 1200 nmol/ml of nitrofurantoin from 30 to 90 min. In control (no nitrofurantoin or α-tocopherol) livers, tissue levels of GSH and PSH were maintained at 78 ± 6% and 94 ± 6% of their initial values, but α-tocopherol levels declined from 34.9 ± 0.6 nmol/g to 26 ± 1.5 nmol/g. In the nitrofurantoin-only livers, GSH and PSH tissue levels fell to 17 ± 10% and 49 ± 6% of their initial levels (P < 0.05, vs. controls). α-Tocopherol levels fell from 31.7 ± 4.8 nmol/g to 17.0 ± 2.8 nmol/g. In the α-tocopherol-supplemented, nitrofurantoin-treated livers, initial tissue levels of α-tocopherol were elevated to 43.8 ± 1.4 nmol/g (P < 0.05), but fell to 26.1 ± 4.2 nmol/g. Their GSH and PSH tissue levels also declined to 7 ± 3% and 56 ± 8% of their initial values (P < 0.05, vs. controls). Thus, it appeared that α-tocopherol supplementation was unable to prevent nitrofurantoin-induced changes in the GSH or PSH status of the liver.