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.

Benzene poisoning, a risk factor for hematological malignancy, is associated with the NQO1 609C-->T mutation and rapid fractional excretion of chlorzoxazone.

Benzene is a ubiquitous occupational hematotoxin and leukemogen, but people vary in their response to this toxic agent. To evaluate the impact of interindividual variation in enzymes that activate (i.e., CYP2E1) and detoxify (i.e., NQO1) benzene and its metabolites, we carried out a case-control study in Shanghai, China, of occupational benzene poisoning (BP; i.e., hematotoxicity), which we show is itself strongly associated with subsequent development of acute nonlymphocytic leukemia and the related myelodysplastic syndromes (relative risk, 70.6; 95% confidence interval, 11.4-439.3). CYP2E1 and NQO1 genotypes were determined by PCR-RFLP, and CYP2E1 enzymatic activity was estimated by the fractional excretion of chlorzoxazone (fe(6-OH)) for 50 cases of BP and 50 controls. Subjects with both a rapid fe(6-OH). and two copies of the NQO1 609C-->T mutation had a 7.6-fold (95% confidence interval, 1.8-31.2) increased risk of BP compared to subjects with a slow fe(6-OH) who carried one or two wild-type NQO1 alleles. In contrast, the CYP2E1 PstI/RsaI polymorphism did not influence BP risk. This is the first report that provides evidence of human susceptibility to benzene-related disease. Further evaluation of susceptibility for hematotoxicity and hematological malignancy among workers with a history of occupational exposure to benzene is warranted.

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.

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

1. Nitrofurantoin is an antimicrobial agent which produces hepatotoxicity caused by 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. Our goal was to determine treatment strategies which could mitigate nitrofurantoin-induced toxicity in the isolated perfused rat liver. We co-infused various agents which blocked early or late events in the progression to toxicity. Tissue levels of glutathione and protein thiols were measured as indicators of the progression to toxicity and lactate dehydrogenase leakage into the perfusate was used as a marker of irreversible cell death. 3. Five treatments significantly (P < 0.05) decreased LDH leakage (reported as thousands of units accumulated in perfusate at 300 min, mean+/-standard error, n = 3-4) when compared to nitrofurantoin alone (274 +/- 37). These treatments were adenosine-2'-monophosphate (120 +/- 53), penicillamine (90 +/- 29), N-(2-mercaptopropionyl)-glycine (120 +/- 49) and bromosulfophthalein with (80 +/- 29) or without 5,5'-difluro-1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraace tic acid (101 +/- 46). Two other treatments, N-acetylcysteine (183 +/- 7) and dithiothreitol (166 +/- 59) delayed the onset of toxicity. Finally, calpeptin (319 +/- 34) which blocks activation of nonlysosomal proteases was ineffective. 4. We concluded that early intervention on the pathway to toxicity was most effective. The strategies detailed here may prove beneficial in treating hepatotoxicity seen following nitrofurantoin therapy.

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.

Comparison of Fe-HBED and Fe-EHPG as hepatobiliary MR contrast agents.

The authors studied the biodisposition and hepatobiliary transport of two potential magnetic resonance imaging contrast agents, the iron (III) chelates of N,N'-bis(2-hydroxybenzyl)ethylene-diamine-N,N'-diacetic acid (HBED) and diastereomeric N,N'-ethylenebis(2-hydroxyphenylglycine) (EHPG). Fecal and urinary excretion (mean +/- standard deviation), respectively, of FE-59 over 7 days in rats given tracer doses of the contrast agents were 67% +/- 2% and 22% +/- 1% for Fe-59-HBED and 22% +/- 2% and 26% +/- 5% for Fe-59-EHPG. In bile duct-cannulated rats given 0.05-mmol/kg doses labeled with Fe-59, 52% +/- 8% of Fe-59 from Fe-59-HBED but only 17% +/- 3% of that from Fe-59-EHPG was excreted into the bile within 90 minutes. Bromosulfophthalein, but not taurocholate or oxyphenonium, was shown to compete with both agents for hepatobiliary transport. Enterohepatic recirculation of both agents was less than 1%.

Alpha-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 alpha-tocopherol might protect protein thiols (PSH) from oxidation, we were interested in determining if alpha-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 alpha-tocopherol succinate. Treated livers were exposed to 1200 nmol/ml of nitrofurantoin from 30 to 90 min. In control (no nitrofurantoin or alpha-tocopherol) livers, tissue levels of GSH and PSH were maintained at 78 +/- 6% and 94 +/- 6% of their initial values, but alpha-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 less than 0.05, vs. controls), alpha-Tocopherol levels fell from 31.7 +/- 4.8 nmol/g to 17.0 +/- 2.8 nmol/g. In the alpha-tocopherol-supplemented, nitrofurantoin-treated livers, initial tissue levels of alpha-tocopherol were elevated to 43.8 +/- 1.4 nmol/g (P less than 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 less than 0.05, vs. controls). Thus, it appeared that alpha-tocopherol supplementation was unable to prevent nitrofurantoin-induced changes in the GSH or PSH status of the liver.

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.

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.

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.