Mitigation of nitrofurantoin-induced toxicity in the perfused rat lung.

1. Nitrofurantoin is an antimicrobial agent which produces pulmonary toxicity via the redox cycling of the nitro group and its radical anion. This futile cycling triggers a complex series of events known collectively as oxidative stress. 2. In the isolated perfused rat lung, nitrofurantoin induced a decrease in tissue levels of glutathione but not protein thiols by the end of the 180 min experiment. There was no decline in tissue levels of angiotensin converting enzyme (a marker of cell disruption). However, edema was extensive as monitored in real time by weight gain (2.71 +/- 0.56 g vs 0.63 +/- 0.53 g in control, P < 0.05, n = 4) and lung mechanical functioning. The edema was matched by an increase in lavage proteins (85 +/- 15 mg vs 16 +/- 9 mg in controls, P < 0.05, n = 4). Electron microscopic examination of tissue indicated that the endothelial cells were detached from the basement membrane which would account for the edema. 3. Co-infusion of penicillamine, N-acetylcysteine or N-(2-mercaptopropionyl)-glycine which can protect tissue from oxidative stress failed to mitigate NFT-induced edema. Allopurinol, an inhibitor of xanthine oxidase and a metal chelator, significantly decreased weight gain but did not prevent the loss of glutathione. These results suggested that allopurinol was not blocking metabolic activation of NFT by xanthine oxidase but scavenging metal cations which can initiate and/or propagate the oxidative stress cascade. 4. We concluded that, in the isolated perfused rat lung, the classic pathway of oxidative stress induced by NFT is interrupted at the stage of GSH loss. These experiments demonstrated that organ function was compromised more than the individual cells. They also suggested that allopurinol may prove beneficial in modulating NFT pulmonary toxicity.

Variability in the disposition of chlorzoxazone.

Chlorzoxazone is 6-hydroxylated by cytochrome P450 2E1 (CYP 2E1), which bioactivates many toxic and carcinogenic molecules. Seventeen volunteers of varying age, ethnicity, and gender received a 250 mg tablet of chlorzoxazone and their blood and urine were sampled frequently for 8 h. V/F = 42 +/- 21 L and CL/F = 412 +/- 120 mL min-1. Comparison of these values with a study by other investigators using a suspension dosage form suggested that relative Ftablet approximately 0.7. The fraction excreted in the urine as 6-hydroxychlorzoxazone (fe,6-OH) was 0.39 +/- 0.20 and that portion of the total CL accounted for by CYP 2E1-mediated metabolism (CL6-OH) was 163 +/- 95 mL min-1. Thus, while V/F and CL/F varied by factors of less than five, fe,6-OH varied 16-fold and CL6-OH varied 28-fold. These results suggested that there was considerable inter-individual variability in the metabolism of chlorzoxazone to 6-hydroxychlorzoxazone. This variability will significantly affect the construction of physiologically based pharmacokinetic models that use the 6-hydroxylation of chlorzoxazone as a marker for an individual's CYP 2E1 phenotype.

Predicting the hepatic clearance of xenobiotics in humans from in vitro data.

Values for Vmax and Km determined during the in vitro metabolism of a xenobiotic to a known metabolite by a specific human isozyme of cytochrome P450 (P450) were used to predict the hepatic clearance (CLH) of the xenobiotic to that metabolite. The calculated CLH values were then compared to literature values of clearance (CL) to the same metabolite obtained during pharmacokinetic studies in humans. For the 6-hydroxylation of chlorzoxazone (P450 2E1) the predicted and actual clearances were 110 +/- 77 mL min-1 and 110 mL min-1, respectively. For the 6 beta-hydroxylation of cortisol, the deethylation of lidocaine (two studies), and the oxidation of nifedipine (all P450 3A3/4) the values were 13 +/- 15 mL min-1 and 13 mL min-1; 758 +/- 282 or 829 +/- 283 mL min-1 and 875 mL min-1; and 284 +/- 176 mL min-1 and 294 mL min-1, respectively. An increase to 72 +/- 25 mL min-1 in the CLH of cortisol to 6 beta-hydroxycortisol was calculated following rifampicin treatment. Finally, the polymorphic nature of the metabolism (P450 2D6) of mexiletine was confirmed. The usefulness of the method and its limitations are discussed.

Pharmacokinetic interaction between benzene metabolites, phenol and hydroquinone, in B6C3F1 mice.

There is strong evidence that metabolites are responsible for adverse effects of benzene. Benzene myelotoxicity, reproduced by coadministering phenol (PH) and hydroquinone (HQ) but not when these benzene metabolites were administered alone, has been postulated to be induced by PH stimulating the myeloperoxidase-mediated oxidation of HQ to the toxic 1,4-benzoquinone in bone marrow. A pharmacokinetic interaction between PH and HQ is also hypothesized to contribute to the observation. Both metabolites are sulfoconjugated and glucuronoconjugated. Sulfoconjugation of phenolic substrates has been shown to approach saturation at high concentrations in rats. Thus, more PH may be converted to HQ and HQ conjugation may be diminished. These effects would increase the amounts of PH and HQ present and result (by further oxidation) in the formation of more 1,4-benzoquinone. To test this hypothesis, we investigated the pharmacokinetics in blood and the recovery of hydroquinone and phenol in urine when the metabolites were administered intraperitoneally alone or in combination at 75 mg/kg each to B6C3F1 mice. The combination resulted in a 2.6-fold increase in the area under the blood concentration-time curve (AUC) of HQ compared to the sum of AUC values observed after administration of each compound alone. The half-life of HQ was also increased from 9 +/- 2 to 15 +/- 3 min. The AUC of PH was increased by a factor of 1.4. The clearance of phenol decreased from 89 +/- 13 ml/min per kilogram when injected alone to 62 +/- 7 ml/min per kilogram after coadministration. A decreased clearance of formation of each conjugate demonstrated that both conjugation pathways were diminished. This interaction may contribute to the observed production of myelotoxicity when these metabolites are coadministered.

Effects of low-density lipoprotein and ethinyl estradiol on cyclosporine metabolism in isolated rat liver perfusions.

The effects of low-density lipoprotein (LDL) on cyclosporine (CyA) metabolism were studied in the isolated perfused rat liver, in a recirculating mode, using Krebs-Ringer buffer in the absence (control perfusion) or presence of LDL (1 microM) (LDL perfusion). In the LDL perfusions, CyA concentrations at all sampling times were about 2-fold higher, whereas the biliary excretion of CyA and measured metabolites (AM1, AM9, AM1c, and AM4N) were all lower than those obtained with the control perfusions. At the end of the perfusion (3 hr), the percentage of total CyA remaining (liver, bile, and perfusate) was significantly higher (76 +/- 1.2% to 85 +/- 2.4%) and the percentage of dose metabolized to AM9 was lower (4.8 +/- 1.2% to 2.4 +/- 0.6%) in the LDL perfusions (N = 4). These results further suggest the inhibitory effects of LDL on CyA uptake, and, thereby, its metabolism as we observed previously in isolated rat hepatocyte studies. Because ethinyl estradiol (EE) is known to increase LDL receptors in rats, we investigated the possible involvement of LDL receptors in transporting CyA into liver cells using rats pretreated with EE (5 mg/kg/day sc for 5 days). The effects of LDL in maintaining CyA perfusate concentrations, and in decreasing biliary excretion of CyA and its metabolites in the EE-treated animals, were in the same direction as those noted in animals without EE, but the differences due to LDL were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Ferrioxamine B derivatives as hepatobiliary contrast agents for magnetic resonance imaging.

Succinyl (SDF), phenylsuccinyl (PSDF), glutaryl (GDF), and phenylglutaryl (PGDF) derivatives of desferrioxamine B (DF) have been synthesized. In rats given the 59Fe(III) chelates of each these ligands at tracer levels, 82-94% of the 59Fe was eliminated within 1-2 days. 59Fe given as DF, SDF, and GDF chelates was excreted primarily in the urine, while nearly 50% of that given as PSDF and PGDF was excreted in the feces. Correspondingly, Fe-DF, Fe-SDF, and Fe-GDF (0.2 mmol/kg) produced early, marked renal, but no gastrointestinal magnetic resonance imaging (MRI) enhancement. Fe-PSDF and Fe-PGDF (0.2 mmol/kg) produced marked and rapid MRI enhancement of the upper small intestine. In animals with cannulated bile ducts, 59Fe from 59Fe-PGDF (carrier added, 0.1 mmol/kg) appeared rapidly in the collected bile, but not in the intestinal contents, proving that the contrast agent reaches the bowel via the bile. These changes in the excretion and MRI enhancement patterns brought about by the presence of a phenyl substituent apparently were not related to changes in lipophilicity or protein binding.

Hepatic transport of the magnetic resonance imaging contrast agent Fe(III)-N-(3-phenylglutaryl)desferrioxamine B.

We have studied the hepatic transport of Fe(III)-N-(3-phenylglutaryl)desferrioxamine B (Fe-PGDF). Using 59Fe-PGDF in biliary cannulated rats we have shown that 32 +/- 2% of an iv bolus dose was excreted into the bile. In animals pretreated with a saturating dose of taurocholate, oxyphenonium, or bromosulfophthalein (BSP), 25 +/- 6%, 23 +/- 1%, or 1.6 +/- 0.8% of the dose, respectively, was excreted into the bile. Magnetic resonance images indicated that BSP blocked uptake of Fe-PGDF by the hepatocytes. Possible enterohepatic recycling of 59Fe-PGDF was investigated in linked rat experiments. All of the material excreted into the bile of the donor, 28 +/- 10% of the dose, was recovered in the gastrointestinal tract of the recipient, but none was found in its bile or urine. These results suggested that uptake of Fe-PGDF by the hepatocytes occurred via the BSP transporter and that no enterohepatic recycling of the contrast agent occurred.

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.

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.

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.

High pressure liquid chromatographic determination of furazolidone in turkey tissue.

A high pressure liquid chromatographic method for determining furazolidone in turkey tissue has been developed. Tissues are ground with methanol and centrifuged. For lower levels of furazolidone, 2--40 ppb, the supernate is evaporated to dryness and redissolved before it is injected onto the liquid chromatographic column. Using a reverse phase column and an ultraviolet absorption detector set at 365 nm, the assay is linear over the concentration range 2--400 ppb with a coefficient of variation of less than 4%. Average recovery from fortified tissues was 96% with a coefficient of variation of 6% at the 50--400 ppb level, and 105% with a coefficient of variation of 11% at the 2--40 ppb level.

Reductive metabolism of nitrofurantoin in the rat.

The reductive metabolism of nitrofurantoin under anaerobic conditions was characterized in various tissues from control, germ-free, and germ-free acclimatized rats. Nitrofurantoin metabolism was highest in homogenates of cecum and colon contents of germ-free acclimatized and control rats, but was absent from those of germ-free animals. Appreciable levels of activity were also present in homogenates of liver and of small intestine walls with lesser rates of metabolism observed in kidney homogenates. The major metabolite of nitrofurantoin, which was isolated and purified by high-pressure liquid chromatography, was identified as 1-[[(3-cyano-1-oxopropyl)-methylene]amino]-2,4-imidazolidinedione. A second, minor metabolite with high-pressure liquid chromatography and ultraviolet absorption characteristics similar to those of 1-[[(5-amino-2-furanyl)methylene]amino]-2,4-imidazolidinedione (aminofurantoin) was detected in cecum and colon contents.

Use of the 295- to 300-nanometer circular dichroism through of ribonucleic acid to study helix winding: effect of acridine orange.

Acridine orange decreases the amplitude of the 295-nm circular dichroism (CD) trough of ribosomal ribonucleic acid (rRNA) where the trough has been related to coil character. Since acridine orange is known from earlier work to intercalate between base pairs of nucleic acids, causing an unwinding of the coil, and our studies show a decrease in the 295-nm CD trough, it appears that CD measurements may be used to observe relative unwinding of rRNA. Under similar solution conditions, melting temperatures with acridine orange indicate no significant change in the stabilization of rRNA structure by acridine orange. Hypochromicity studies show no increase in the percent base pairing in rRNA when 0.1 M tris(hydroxymethyl)aminomethane (pH 7.6) with 1.35 M KCl is used. These results indicate that CD changes in the amplitude of the 295-nm trough of rRNA are related to helix winding in rRNA.