Xenobiotic metabolism in human alveolar type II cells isolated by centrifugal elutriation and density gradient centrifugation.

Alveolar type II cells were isolated from five human lung specimens obtained during resection or lobectomy and enriched to 63-85% purity. Digestion with Sigma protease type XIV followed by centrifugal elutriation and Percoll density gradient centrifugation yielded 1.2 +/- 0.4 X 10(6) cells/g lung in the type II cell fractions. The activities of some enzymes involved in the metabolism of xenobiotics were determined in these freshly isolated type II cells and compared with activities in alveolar macrophages and fractions of unseparated cells from the same tissue samples. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was similar in the three cell fractions from all five patients (18-29 nmol/mg protein/min). An antibody to rabbit reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase inhibited reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reduction as much as 70% in microsomal preparations of the isolated human pulmonary cells, although this same antibody barely reacted with microsomes of the human cells in a Western blot assay. Epoxide hydrolase activity was highest in the alveolar type II cells (1.08 +/- 0.17 nmol/mg protein/min). This activity was 6 times higher than in the alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarin deethylase activity, a cytochrome P-450-dependent pathway, was low or undetectable in the three cell fractions. Trace amounts of 7-ethoxyresorufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detected in microsomes of the isolated human cells, even though a polycyclic hydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxyresorufin (form 6 in rabbits) was not detected immunochemically.

High frequency and heterogeneous distribution of p53 mutations in aflatoxin B1-induced mouse lung tumors.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human lung cancers. However, p53 mutations are more rarely detected in chemically induced mouse lung tumors. In this study, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with aflatoxin B1 (AFB1; 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6-14 months after dosing, mice were killed, and tumors were collected. A total of 71 AFB1-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB1-induced tumors, but the pattern was highly heterogeneous. In approximately 73% of the positively stained tumors, fewer than 5% of cells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery of the tumors in many cases. Single-strand conformational polymorphism analysis of the evolutionarily conserved regions of the p53 gene (exons 5-8) from AFB1-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutations could not be confirmed by direct sequence analysis because bands representing putative mutations appeared only weakly on autoradiograms. This was presumably due to the heterogeneous nature of the DNA analyzed. Single-strand conformational polymorphism analysis of DNA from laser capture microdissected cells of paraffin-embedded AFB1-induced tumor tissue sections stained for p53 produced banding patterns consistent with point mutations in 18 of 30 (60%) DNA samples. Direct sequencing of the microdissected samples revealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mutations found in microdissected regions from adenomas and carcinomas, 9 were G:C-->A:T transitions, 11 were A:T-->G:C transitions, and 5 were transversions (2 G:C-->T:A, 2 T:A-->A:T, and 1 A:T-->C:G), whereas 1 deletion mutation was identified. The concordance between immunostaining and molecular detection of p53 alterations was 72% when laser capture microdissection was used versus 17% based on whole tumor analysis. The high mutation frequency and heterogeneous staining pattern suggest that p53 mutations occur relatively late in AFB1-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung tumors.

Human lung microsomal cytochrome P4501A1 (CYP1A1) activities: impact of smoking status and CYP1A1, aryl hydrocarbon receptor, and glutathione S-transferase M1 genetic polymorphisms.

There are numerous conflicting epidemiological studies addressing correlations between cytochrome P450 1A1 (CYP1A1) genetic polymorphisms and lung cancer susceptibility, with associations plausibly linked to alterations in carcinogen bioactivation. Similarly, correlations between aryl hydrocarbon receptor gene (AHR) codon 554 genotype and CYP1A1 inducibility are controversial. The objective of this study was to determine whether smoking status, and CYP1A1, AHR, and glutathione S-transferase M1 gene (GSTM1) polymorphisms correlate with altered CYP1A1 activities. Lung microsomal CYP1A1-catalyzed 7-ethoxyresorufin O-dealkylation (EROD) activities were much higher in tissues from current smokers (n = 46) than in those from non-/former smokers (n = 24; 12.11 +/- 13.46 and 0.77 +/- 1.74 pmol/min/mg protein, respectively, mean +/- SD; P < 0.05). However, EROD activities in lung microsomes from current smokers CYP1A1*1/1 (n = 33) and heterozygous MspI variant CYP1A1*1/2A (n = 10) were not significantly different (12.23 +/- 13.48 and 8.23 +/- 9.76 pmol/min/mg protein, respectively, P > 0.05). Three current smokers were heterozygous variant CYP1A1*1/2B (possessing both *2A and *2C alleles), and exhibited activities similar to individuals CYP1A*1/1. One current smoker was heterozygous variant CYP1A1*4 and exhibited activities comparable with individuals CYP1A1*1/1 at that locus. EROD activities in microsomes from current smokers AHR(554)Arg/Arg (n = 41) and heterozygous variant AHR(554)Arg/Lys (n = 5) were not significantly different (12.13 +/- 13.56 and 12.01 +/- 14.23 pmol/min/mg protein, respectively; P > 0.05). Furthermore, microsomal EROD activities from current smokers with the GSTM1-null genotype (n = 28) were not significantly different from those (n = 18) carrying at least one copy of GSTM1 (12.61 +/- 14.24 and 11.34 +/- 12.53 pmol/min/mg protein, respectively; P > 0.05). Additionally, when genotypic combinations of CYP1A1, AHR, and GSTM1 were assessed, there were no significant effects on EROD activity. On the basis of microsomal enzyme activities from heterozygotes, CYP1A1*1/2A, CYP1A1*1/2B, CYP1A1*1/4, and AHR(554) Arg/Lys variants do not appear to significantly affect CYP1A1 activities in human lung, and we observed no association between CYP1A1 activity and the GSTM1-null polymorphism.

In vitro metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by lung cells isolated from Syrian golden hamster.

The nicotine derived N-nitrosamine, 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK), is a potent respiratory carcinogen in the Syrian golden hamster. The in vitro metabolism of NNK by three enriched lung cell populations were compared. Clara cells had more than ten times higher capacity to activate NNK by alpha-carbon hydroxylation than alveolar macrophages and fibroblasts. In the former cell types, levels of deactivation of NNK by pyridine N-oxidation were ten times lower than those of alpha-carbon hydroxylation. In alveolar macrophages, carbonyl reduction was the major metabolic pathway. Our results suggest that DNA damages induced by NNK in hamster lung is more likely to occur in Clara cells than in macrophages or fibroblasts.

Modulation of aflatoxin B1 biotransformation in rabbit pulmonary and hepatic microsomes.

Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin that requires activation to the corresponding 8,9-epoxide for activity. In addition to being present in foodstuffs, AFB1 can contaminate respirable grain dusts and thus the respiratory system is a potential target for carcinogenesis. In the present study, we have investigated the role of polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 in the pulmonary and hepatic microsomal activation ([3H]AFB1-DNA binding) and detoxification ([3H]AFM1 and [3H]AFQ1 formation) of [3H]AFB1. In rabbit lung microsomes, the apparent Vmax for [3H]AFM1 formation was increased significantly when values were expressed per mg microsomal protein or per nmol P-450 present. In liver microsomes, the apparent Vmax for DNA binding and [3H]AFM1 formation were increased by beta-naphthoflavone (BNF) treatment (to 2.3 and 3.3 times control, respectively) when expressed per mg protein, but when expressed per nmol P-450, only AFM1 formation was significantly increased. The apparent Km values for both these reactions were unaffected. The apparent Vmax for [3H]AFQ1 formation was not affected by BNF treatment, but the apparent Km was increased to 4.5 times control. Boiling of microsomes or omitting the NADPH-generating system decreased DNA binding, AFM1 formation and AFQ1 formation by 89-97%, while addition of 1.0 mM SKF-525A inhibited these reactions by 46-57%. Addition of 1.0 mM alpha-naphthoflavone (ANF) had no effect on the biotransformation of [3H]AFB1 in lung microsomes of control rabbits, but significantly decreased AFM1 formation (by 31%) in lung microsomes from BNF-treated animals (other reactions were unaffected). In liver microsomes from BNF treated rabbits, 1.0 mM ANF inhibited DNA binding of [3H]AFB1 by 68%, while there was no effect in control microsomes. ANF significantly inhibited AFM1 formation in liver microsomes from both control and BNF-treated animals (by 87-97% and 67-78% at 1.0 mM and 2.0 microM, respectively), but had no effect on AFQ1 formation in liver microsomes from animals in either treatment group. These results indicate an important role for the 1A subclass of P-450 isozymes in the biotransformation of AFB1 to AFM1 in rabbit lung and liver, and a minor role in AFB1 activation in liver.

Increased acetaminophen-induced hepatotoxicity after chronic ethanol consumption in mice.

The effect of chronic ethanol consumption on acetaminophen (200, 400, and 600 mg/kg) toxicity was determined by maintaining mice for 10 days on diets consisting of chow and one of the following drinking solutions: 10% ethanol + 10% sucrose, 8% sucrose, or tap water. Toxicity as manifested by mortality, liver enlargement, and liver congestion was greatest in the ethanol-treated group. We suggest that the greater mortality was a result of the increased liver congestion and consequent hypovolemia. Despite the increased levels of cytochrome(s) P-450, covalent binding of [3H]acetaminophen reactive metabolite(s) to liver protein was not higher in ethanol-treated animals. This can be explained by the higher initial glutathione concentration and/or ability to replenish glutathione in the ethanol-treated group. We suggest that the enhancement of acetaminophen toxicity by ethanol is the result of an effect of ethanol on hepatocyte membranes which renders the cells more susceptible to toxic injury.

Effects of vitamin E on cytotoxicity of amiodarone and N-desethylamiodarone in isolated hamster lung cells.

Amiodarone (AM) is a potent and efficacious antidysrhythmic agent that can cause potentially life-threatening pulmonary fibrosis. Vitamin E has been demonstrated to decrease AM-induced pulmonary fibrosis in vivo in hamsters. In the present in vitro study, we investigated the effects of vitamin E on cell death induced by AM and its primary metabolite, N-desethylamiodarone (DEA), in freshly isolated hamster lung cells. Following incubation for 24 or 36 h, 300 microM vitamin E decreased (P<0.05) 100 microM AM-induced cytotoxicity (0.5% trypan blue uptake) in alveolar macrophages by 11.7+/-3% or 21.4+/-12%, respectively, but did not decrease cytotoxicity in fractions enriched with alveolar type II cells or non-ciliated bronchiolar epithelial (Clara cells) or in isolated unseparated cells (cell digest). Vitamin E had no effect on 50 microM DEA-induced cytotoxicity. Vitamin E did not alter cellular levels of AM or DEA in any cell fraction. Lipid peroxidation (assessed by isoprostane formation) was increased (P<0.05) in cell digest, alveolar type II cell and Clara cell enriched fractions incubated with 500 microM carbon tetrachloride (CCl(4)) for 4 h but not in enriched fractions of cells exposed to 100 microM AM or 50 microM DEA. No AM-induced loss of viability was observed at this time point, but DEA decreased (P<0.05) Clara cell viability by approximately 25%. These results demonstrate cell type selective protection against AM-induced cytotoxicity by vitamin E, and suggest that lipid peroxidation does not initiate AM- or DEA-induced cytotoxicity in isolated hamster lung cells.

Acetaminophen-induced hypothermia in mice: evidence for a central action of the parent compound.

Pretreatment of mice with phenobarbital, an inducer of oxidative drug metabolism, had no effect on the early hypothermic effect of a toxic dose of acetaminophen, while pretreatment with metyrapone, SKF-525A, or piperonyl butoxide (inhibitors of mixed-function oxidase) enhanced the hypothermia. In mice treated with acetaminophen alone, brain parent drug levels correlated with the degree of hypothermia, while liver drug levels did not. Also, intracerebroventricular injection of acetaminophen resulted in significant hypothermia within 20 min. These results indicate that the early hypothermia caused by acetaminophen in mice is due to the parent drug, not to its toxic reactive metabolite, and that the effect is mediated centrally. The observation that piperonyl butoxide and SKF-525A themselves caused significant hypothermia indicates that the use of these compounds should be avoided when body temperature is being followed in drug metabolism experiments.

Furosemide toxicity in isolated mouse hepatocyte suspensions.

Incubation of freshly isolated mouse hepatocytes with 0.5 or 1.0 mM furosemide caused a depletion of cellular acid soluble sulfhydryls to approximately 20-30% of control over the course of 4.5 h. The depletion was accompanied by a reduction in cell viability (indicated by the lactate dehydrogenase latency test) which was significant (P less than 0.05) for 0.5 mM but not for 1.0 mM furosemide at 4.5 h. Ultrastructurally, 0.5 or 1.0 mM furosemide caused cytoplasmic changes including loss of glycogen, disaggregation of polyribosomes, vesiculation of endoplasmic reticulum, and occasional appearance of lamellar bodies consisting of concentric arrays of paired smooth membranes. These concentrations of furosemide also caused cell surface changes, including loss of microvilli, development of an irregular shape compared to the spherical appearance of untreated hepatocytes, and the development of occasional blebs. The appearance of pale staining hydropic cells was indicative of the final stages of cell death. N-Acetylcysteine (6.0 mM) was effective at preventing the depletion of soluble sulfhydryls, the loss of viability, and the ultrastructural effects of 0.5 or 1.0 mM furosemide, suggesting a role for soluble sulfhydryls in the pathogenesis of furosemide hepatotoxicity.

Effects of dietary vitamin E supplementation on pulmonary morphology and collagen deposition in amiodarone- and vehicle-treated hamsters.

Amiodarone (AM) is a potent antidysrhythmic agent that is limited in clinical use by its adverse effects, including potentially life-threatening AM-induced pulmonary toxicity (AIPT). The present study tested the ability of dietary supplementation with vitamin E (500 IU d,1-alpha-tocopherol acetate/kg chow) to protect against pulmonary damage following intratracheal administration of AM (1.83 micromol) to the male golden Syrian hamster. At 21 days post-dosing, animals treated with AM had increased lung hydroxyproline content and histological disease index values compared to control (P < 0.05), which were indicative of fibrosis. Dietary vitamin E supplementation for 6 weeks resulted in a 234% increase in lung vitamin E content at the time of AM dosing, and maintenance on the diet prevented AM-induced elevation of hydroxyproline content and disease index 21 days post-dosing. Dietary vitamin E supplementation also decreased hydroxyproline content and disease index values in hamsters treated intratracheally with distilled water, the AM vehicle. These results demonstrate a protective role for vitamin E in an in vivo model of AIPT, and suggest that this antioxidant may have non-specific antifibrotic effects in the lung.

Resistance of the hamster to amiodarone-induced pulmonary toxicity following repeated intraperitoneal administration.

Amiodarone is an effective antidysrhythmic agent, restricted in use by the development of pulmonary toxicity. Several in vivo animal models have been used to study amiodarone-induced pulmonary toxicity. Intratracheal administration of amiodarone to the hamster has been used as a model for the critical amiodarone-induced pulmonary fibrosis (AIPF). In order to investigate the cellular mechanism of human AIPF, which occurs following oral or intravenous administration, an animal model of AIPF resulting from systemic administration of the drug would appear to be preferable. We have evaluated pulmonary toxicity following repeated intraperitoneal amiodarone administration to the hamster. Intraperitoneal treatment of hamsters for 1, 4, or 7 weeks with amiodarone (100 mg/kg/day) did not lead to pulmonary toxicity based on wet lung weight, hydroxyproline content, or histological examination. Furthermore, when comparing 1- and 7-week treatment groups, there was no pulmonary accumulation of either amiodarone or desethylamiodarone beyond levels found at 1 week. Therefore, failure to develop pulmonary toxicity may be due to an inability to accumulate sufficient amiodarone and/or desethylamiodarone. Intratracheal administration of amiodarone to rodents remains the only in vivo animal model for studying the mechanism(s) of AIPF.

Amiodarone-induced disruption of hamster lung and liver mitochondrial function: lack of association with thiobarbituric acid-reactive substance production.

Amiodarone (AM) is an efficacious antidysrhythmic agent that is limited clinically by numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT) due to the potential for mortality. Mitochondrial alterations and free radicals have been implicated in the etiology of AM-induced toxicities, including AIPT. Isolated hamster lung and liver mitochondria were assessed for AM-induced effects on respiration, membrane potential, and lipid peroxidation. AM (50-400 microM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 microM) resulting in a subsequent inhibition. This biphasic effect of AM (200 microM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50-400 microM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 microM AM decreased lung mitochondrial membrane potential (p < 0.05), while a concentration-dependent (50-200 microM) decrease of membrane potential was observed with liver mitochondria exposed to AM (p < 0.05). Formation of thiobarbituric acid-reactive substances (TBARS) was not altered by AM (50-400 microM) in incubations lasting up to 1 h. These results indicate that lipid peroxidation, as indicated by levels of TBARS, does not play a role in AM-induced alterations in mitochondrial respiration and membrane potential.

The 1995 Pharmacological Society of Canada Merck Frosst Award. Cellular and molecular targets in pulmonary chemical carcinogenesis: studies with aflatoxin B1.

Although most notorious as a liver carcinogen, the mycotoxin aflatoxin B1 targets other tissues as well, including those of the respiratory system. Because the biotransformation of aflatoxin B1 to toxic and nontoxic metabolites has been fairly well characterized, it serves as a useful and relevant model carcinogen for studying the biochemical (i.e., balance between bioactivation and detoxification) and molecular (i.e., mutations in target genes) mechanisms of pulmonary chemical carcinogenesis. Because of the cellular diversity of the lung, it is of particular interest to assess these processes in different lung cell types, if we are to identify the target cells for carcinogen action. This review summarizes studies that have been aimed at identifying the basis for susceptibility of the lung to aflatoxin B1.

Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas.

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 micron diameter fraction believed to be composed largely of basal cells, and a 15 micron diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 micron cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 +/- 2.7% ciliated cells with 6.5 +/- 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 +/- 2.1%) and Clara cells (27.0 +/- 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 +/- 0.09 X 10(6) cells/trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 micron cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 +/- 66 pmol/min/mg protein or 51.2 +/- 20.5 pmol/min/10(6) cells for 7-ethoxycoumarin deethylase and 31.7 +/- 15.4 pmol/min/mg protein or 10.5 +/- 4.8 pmol/min/10(6) cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.

Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic lipoxygenase was significantly lower than those for PHS and P450. Hepatic half-maximal binding concentrations for PHS and P450 were similar, though P450 had a much higher maximum rate than PHS and lipoxygenases. These data suggest that lipoxygenase-catalyzed AFB1 activation can occur at low AFB1 concentrations. This may be important in view of human exposure to low AFB1 concentrations and predominant lipoxygenase activity in human airway epithelial cells. When expressed per gram of tissue, renal and hepatic PHS activities and renal lipoxygenase activities for AFB1 activation were similar, and higher than the activity of pulmonary PHS, while pulmonary PHS activity for the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was similar to that in liver and lower than that in kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

Ki-ras activation in lung cells isolated from AC3F1 (A/J x C3H/HeJ) mice after treatment with aflatoxin B1.

Lung cells isolated from AC3F1 (A/J x C3H/HeJ) mice 7 wk after treatment with the carcinogenic mycotoxin aflatoxin B1 (AFB1) were examined for point mutations in the Ki-ras oncogene. Ki-ras mutant allele frequencies in fractions enriched with nonciliated bronchiolar epithelial (Clara) cells were consistently higher than in alveolar type II cell fractions. Mutant alleles were undetectable or minimal in macrophage- and polymorphonuclear leukocyte-enriched fractions. In cells from vehicle (dimethyl sulfoxide)-treated mice, small proportions of mutant Ki-ras alleles were found in the Clara cell-enriched fraction but not in other cell fractions. The results indicated that Clara cells are particularly susceptible to AFB1-induced Ki-ras mutation, an early event in AFB1-induced mouse lung tumorigenesis.

Biotransformation of xenobiotics in individual rabbit hepatocytes. Application of microspectrofluorometry.

We have investigated several methods for determining rates of xenobiotic biotransformation in individual rabbit hepatocytes by microspectrofluorometry. Experiments designed to measure monooxygenase activity by following fluorescent product formation (i.e. 7-ethoxycoumarin deethylase or oxidation of the fluorescein derivatives ethoxyfluorescein ethyl ester and 5- and 6-ethoxycarbonyl ethoxyfluorescein ethyl ester) demonstrated that the fluorescent products were released from cells to the surrounding media. Thus, metabolically active cells probably had underestimated activities, and inactive cells could accumulate fluorescent product and appear metabolically active. We also utilized benzo(a)pyrene (BP) as a substrate in our system. By selecting appropriate wavelengths (370 nm excitation, 407 nm emission), it was possible to selectively monitor BP disappearance in single cells. In hepatocytes from rabbits, disappearance of fluorescence was observed after a 10-min incubation at 37 degrees C with 1-40 microM BP followed by a wash to remove extracellular substrate. The decrease in fluorescence followed apparent first-order kinetics. Measurement of extracellular fluorescence indicated that the substrate did not leave the hepatocytes. Furthermore, in both control and beta-naphthoflavone-treated rabbits, the observed fluorescence changes in the cells were inhibited by the presence of equimolar concentrations of ellipticine or alpha-naphthoflavone, inhibitors of BP metabolism. Storage of hepatocytes from untreated animals on ice for 3.5 hr did not affect rate constant values in cells with a normal appearance, whereas those lacking refringent edges or having numerous cytoplasmic blebs demonstrated greatly reduced (up to 88% loss) activities compared with normal, freshly isolated cells. Potential applications of this system (e.g. to other cell types) are discussed.

Mechanisms of aflatoxin B1 lung tumorigenesis.

Although aflatoxin B1 (AFB1) is best known as a hepatocarcinogen, the respiratory system can also be a target of this mycotoxin. In isolated lung cells from rabbits and mice, AFB1 is bioactivated by cytochromes P450, primarily in nonciliated bronchiolar epithelial (Clara) cells. However, mutagenesis experiments suggest that the DNA-binding AFB1 epoxide metabolite can leave the cells of origin, and potentially interact with other cell types. Consistent with DNA adduct studies, AFB1-induced AC3F1 mouse lung tumors contain point mutations at guanine residues in K-ras, with the anticipated bias for the A/J allele. Furthermore, following AFB1 treatment but prior to tumor development, K-ras mutations occur preferentially in mouse Clara cells. However, in contrast to findings with other carcinogens, AFB1-induced mouse lung tumors demonstrate frequent, but heterogeneously distributed, overexpression of p53 protein as well as p53 point mutations, suggesting a carcinogen-specific response. Unlike lung tissue from mice and rabbits, human peripheral lung bioactivates AFB1 primarily by prostaglandin H synthase--and/or lipoxygenase-catalyzed cooxidation, with activity concentrated in macrophages. In addition, although glutathione S-transferase M1-1 has high specific activity for AFB1 epoxide conjugation, lung tissues from GSTM1-null individuals do not demonstrate diminished rates of conjugation, compared to tissues from GSTM1-positive individuals. In summary, AFB1 tumorigenesis in mice demonstrates unique properties, and processes of bioactivation show significant species differences.

Investigation of the role of oxidative stress in amiodarone-induced pulmonary toxicity in the hamster.

Associated with amiodarone (AM) therapy is pneumonitis, which may progress to life-threatening pulmonary fibrosis. Although the etiology of amiodarone-induced pulmonary toxicity (AIPT) is unknown, a role for direct toxicity by oxidative stress has been proposed. We have used a single intratracheal administration of AM (1.8 mg (2.64 mumol)) to male golden Syrian hamsters to investigate the role of oxidative stress in AIPT. The antioxidant capacity of the lung was assessed following AM administration by evaluating glutathione status and antioxidant enzyme activities. The efficacy of treatment with the antioxidant agents butylated hydroxyanisole, diallyl sulfide, and N-acetylcysteine, in attenuation of AM-induced pulmonary fibrosis was also investigated. AM significantly (p < 0.05) increased the ratio of oxidized to total lung glutathione both 30 min (control, 0.93 +/- 0.23%; AM, 2.06 +/- 0.26%) and 120 min (control, 0.90 +/- 0.21%; AM, 3.58 +/- 1.34%) post administration. AM also increased activities of glutathione reductase (by 89%) 3 days post administration, and glutathione peroxidase (by 110 and 45%, respectively) and total superoxide dismutase (by 58 and 35%, respectively) both 3 and 7 days post administration. However, treatment of hamsters with butylated hydroxyanisole (150 mg.kg-1.day-1 s.c.) or diallyl sulfide (200 mg.kg-1.day-1, p.o.) for 3 days prior to AM, or treatment with N-acetylcysteine (10 mg intratracheally) 10 min prior to AM had no effect on pulmonary fibrosis 21 days post treatment, as determined by lung wet weight and hydroxyproline content, and had inconsistent effects on histologically determined disease index.(ABSTRACT TRUNCATED AT 250 WORDS)