Dietary zinc supplementation inhibits NFkappaB activation and protects against chemically induced diabetes in CD1 mice.

Zinc status in patients with Type I diabetes is significantly lower than healthy controls. Whether zinc supplementation can prevent the onset of Type I diabetes is unknown. Recent studies have suggested that the generation of reactive oxygen species (ROS) is a cause of beta cell death leading to Type I diabetes. In addition, we found that activation of NFkappaB (a ROS-sensitive transcription factor that regulates immune responses) may be the key cellular process that bridges oxidative stress and the death of beta cells. Zinc is a known antioxidant in the immune system. Therefore, this study is designed to test whether an increase in dietary zinc can prevent the onset of Type I diabetes by blocking NFkappaB activation in the pancreas. The results show that high zinc intake significantly reduced the severity of Type I diabetes (based on hyperglycemia, insulin level, and islet morphology) in alloxan and streptozotocin-induced diabetic models. Zinc supplementation also inhibited NFkappaB activation and decreased the expression of inducible NO synthase, a downstream target gene of NFkappaB. It is concluded that zinc supplementation can significantly inhibit the development of Type I diabetes. The ability of zinc to modulate NFkappaB activation in the diabetogenic pathway may be the key mechanism for zinc's protective effect. Inhibition of the NFkappaB pathway may prove to be an important criterion for choosing nutritional strategies for Type I diabetes prevention.

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia.

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.

Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase.

Conjugated linoleic acid (CLA; 18:2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; delta9,12-18:2). Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 microM) on elongation of LA (25 microM) to eicosadienoic acid (EDA; delta11,14-20:2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 microM to the media, were elongated to conjugated FDA (c11,t13- and t12,c14-20:2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on delta6-desaturation as well as elongation of LA which results in a decrease in long-chain n-6 fatty acids and consequently the eicosanoid synthesis.

Evaluation of the ability of orally administered aspirin to mitigate effects of 3-methylindole in feedlot cattle.

OBJECTIVE: To evaluate the ability of orally administered aspirin to mitigate 3-methylindole (3MI)-induced respiratory tract disease and reduced rate of gain in feedlot cattle. ANIMALS: 244 beef cattle. PROCEDURE: In a masked, randomized, controlled field trial, calves were untreated (controls) or received a single orally administered dose of aspirin (31.2 g) on entry into a feedlot. Serum 3MI concentrations were measured on days 0, 3, and 6. Rumen 3MI concentration was measured on day 3. Cattle were observed daily for clinical signs of respiratory tract disease. Lungs were evaluated at slaughter for gross pulmonary lesions. RESULTS: Mean daily gain (MDG) in cattle treated with aspirin, compared with control cattle, was 0.06 kg greater in the backgrounding unit and 0.03 kg greater for the overall feeding period. Neither serum nor rumen 3MI concentrations appeared to modify this effect. Cattle treated with aspirin were more likely to be treated for respiratory tract disease. Mortality rate, gross pulmonary lesions, and serum and rumen 3MI concentrations were similar between groups. Increased rumen 3MI concentration was associated with a small difference in risk of lung fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Cattle given a single orally administered dose of aspirin on feedlot entry had higher MDG in the backgrounding unit and for the overall feeding period, but this finding could not be attributed to mitigation of effects of 3MI. This may have been influenced by low peak 3MI production and slow rates of gain.

Effects of 3-methylindole production and immunity against bovine respiratory syncytial virus on development of respiratory tract disease and rate of gain of feedlot cattle.

OBJECTIVE: To determine whether immunity against bovine respiratory syncytial virus (BRSV) mitigates the effects of 3-methylindole (3MI) on occurrence of bovine respiratory tract disease (BRD) and rate of gain in feedlot cattle. ANIMALS: 254 mixed-breed beef cattle. PROCEDURE: Cattle were randomly assigned to 1 of 3 groups at the time of arrival at the feedlot. One group was vaccinated with an inactivated BRSV vaccine, another was vaccinated with a modified-live BRSV vaccine, and the third was maintained as unvaccinated control cattle. On days 0 and 28, serum BRSV antibody concentrations were measured, using serum neutralizing and ELISA techniques. Serum 3MI concentrations were measured at feedlot arrival and 3 days later. Cattle were monitored for development of BRD. At slaughter, lungs were evaluated grossly for chronic lesions. RESULTS: Higher serum 3MI concentrations early in the feeding period were associated with lower mean daily gain. Control cattle were more likely to be treated for BRD after day 3, compared with cattle vaccinated with the modified-live BRSV vaccine. Humoral immunity against BRSV did not appear to modify the effect of 3MI on development of BRD or mean daily gain. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that abrogating the effects of 3MI and BRSV infection may improve the health and growth performance of feedlot cattle. However, in this study, immunity against BRSV did not appear to protect against the potential synergism between 3MI and BRSV infection, possibly because of the slow rates of gain of cattle included in the study or timing of sample collection.

Alpha-phenyl-tert-butylnitrone (PBN) inhibits NFkappaB activation offering protection against chemically induced diabetes.

Alpha-phenyl-tert-butylnitrone (PBN) is an effective spin trapping agent by reacting with and stabilizing free radical species. Reactive oxygen species (ROS) have been implicated in pancreatic beta cell death and the development of insulin-dependent diabetes mellitus (IDDM). We speculate that treatment with the PBN, will protect against diabetes development in two distinct chemically induced models for IDDM. Pretreatment with PBN (150 mg/kg ip) significantly reduced the severity of hyperglycemia in both alloxan- and streptozotocin (STZ) induced diabetes. To determine the mechanism by which PBN prevents hyperglycemia, we examined the ability of PBN to inhibit NFkappaB activation and to stabilize alloxan- and STZ-induced radicals. Both alloxan and STZ induced NFkappaB activation in the pancreas 30 min after their injection (50 mg/kg iv). PBN pretreatment inhibited both alloxan- and STZ-induced activation of NFkappaB and nitric oxide production. EPR studies showed that PBN could effectively trap alloxan-induced free radicals. It is clear that PBN can inhibit NFkappaB activation in the pancreas and reduce hyperglycemia in two distinct diabetogenic compounds. This research indicates that NFkappaB activation may be a key signal leading to beta cell death and IDDM. Understanding the cellular pathways leading to beta cell death may help in developing effective preventive or therapeutic targets for IDDM.

Zinc deficiency exacerbates loss in blood-brain barrier integrity induced by hyperoxia measured by dynamic MRI.

Using dynamic Magnetic Resonance Imaging (dMRI), blood-brain barrier (BBB) permeability (k(PSrho)) and tissue interstitial leakage space (v(e)) were evaluated in zinc-deficient (ZnDF) male weanling Wistar rats following 3 days exposure to hyperoxia (85% O2). Temporal monitoring of T1-weighted MR image changes, following a bolus intravenous injection of gadolinium-DTPA, allowed estimation of BBB integrity. Three-day exposure of hyperoxia caused a marginal loss of BBB integrity, reflected in a slight increase in kPSrho and v(e), observed in both the animals fed adequate zinc (ZnAL) and pair-fed controls (ZnPF). However, zinc deficiency resulted in a significant increase in both kPSrho and v(e), indicating a severely disturbed BBB. In addition MR-visible free water was elevated in ZnDF brains following hyperoxia treatment indicating that a loss of BBB integrity may be associated with neuronal edema. The diminished BBB integrity may be free-radical mediated as the ratio of oxidized to reduced glutathione (GSSG:GSH) was significantly elevated.

Antioxidants, NFkappaB activation, and diabetogenesis.

Although many risk factors can trigger the development of insulin-dependent diabetes (IDDM), it is likely that reactive oxygen species (ROS) play a central role in beta-cell death and disease progression. This review will focus on the role of antioxidant defense systems in the susceptibility to IDDM and on ROS as cellular messengers that regulate the expression of genes leading to beta-cell death. Accumulating evidence indicates that increased antioxidant defense systems reduce the susceptibility to IDDM in animal models or in human study. It is suggested that pancreas-specific ROS productions play a critical role in signaling the cellular autoimmune/inflammatory response by activating the transcription factor, NFkappaB. Various diabetogenic factors may lead to an increase in ROS production, which activates the redox-sensitive NFkappaB. This may be the initial event for the expression of cytokines and chemotactic agents involved in the autoimmune/inflammatory response. It is believed that this cascade results in a cyclic amplification of ROS and eventually leads to apoptosis and/or necrosis of beta cells. The specificity of antioxidants to inhibit NFkappaB activation and the hyperglycemic response emphasizes the importance of selectivity in antioxidant therapy. Research in this area will contribute significantly to our understanding of the cellular and mechanistic role of ROS in the etiology of IDDM and will lead to the development of better prevention strategies.

Transcriptional induction of metallothionein-I and -II genes in the livers of Cu,Zn-superoxide dismutase knockout mice.

The levels of metallothionein-I and -II (MT-I and MT-II) mRNAs were elevated (10- to 12-fold), specifically in the livers of mice with homozygous deletion of the gene for Cu,Zn-SOD (Sod1-/-), the enzyme that catalyzes the removal of O(-)(2). The induction of MT mRNA occurred primarily at the level of transcription. In vivo genomic footprinting of the MT-I promoter region revealed distinctive footprinting at MRE-d, MRE-c, and MLTF/ARE sites in the livers of knockout mice. MTF-1, the key factor responsible for the heavy-metal and oxidative stress-induced expression of the MT-I gene, was activated 3-fold in the nuclear extract from the livers of Cu,Zn-SOD null mice. Because metallothioneins are potent scavengers of reactive oxygen species and protect cells from oxidative stress, the apparent normal characteristics of the mice with the disrupted Cu, Zn-SOD gene are probably due to overexpression of MT-I and MT-II in the livers of these animals.

Supplementation of N-acetylcysteine inhibits NFkappaB activation and protects against alloxan-induced diabetes in CD-1 mice.

Reactive oxygen species (ROS) are involved in the destruction of pancreatic beta cells and the development of insulin-dependent diabetes mellitus (IDDM). However, the cellular mechanism responsible for beta cell death is still unclear. We hypothesize that activation of NFkappaB by ROS is the key cellular signal in initiating a cascade of events leading to beta cell death. Thus, enhancement of pancreatic GSH, a known antioxidant and key regulator of NF-kappaB, should protect against IDDM. Weanling CD1 mice (n=5) were injected with alloxan (50 mg/kg i.v.) to induce IDDM. Using EPR spin trapping techniques, we demonstrated that alloxan generated ROS in the pancreas 15 min after administration. Activation of NFkappaB in pancreatic nuclear extracts was observed 30 min after alloxan injection, as assessed by an electrophoretic mobility shift assay. Fasting blood glucose levels were monitored for 14 days. Supplementation with N-acetylcysteine (NAC, 500 mg/kg), a GSH precursor, inhibited alloxan-induced NFkappaB activation and reduced hyperglycemia. Thus, NFkappaB activation by ROS may initiate a sequence of events leading to IDDM. Inhibition of NF-kappaB activation by NAC attenuated the severity of IDDM. This research will contribute to the understanding of the etiology of IDDM and may lead to the development of better strategies for disease prevention.

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.

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.

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.

Effect of oxidative stress on brain damage detected by MRI and in vivo 31P-NMR.

The brain is susceptible to oxidative stress. This is due to the high content of polyunsaturated fatty acids, high rate of oxygen consumption, regional high concentrations of iron, and relatively low antioxidant capacity. These factors may predispose the premature infant to brain damage. Brain damage may be due to: 1. Brief anoxia followed by hyperoxia (mimics parturition oxidative stress); or 2. Prolonged exposure to hyperoxia (mimics oxidative stress from postpartum maintenance in a hyperoxic environment). We have developed two animal models to examine these forms of oxidative stress on the brains of rats. In Model I rats were exposed to brief anoxic anoxia (100% N2) followed by hyperoxia (100% O2). Using T2-weighted Magnetic Resonance Imaging (MRI) brain intensity decreased following the treatment suggesting water loss or free radical production. In vivo 1H-NMR showed brain water content appeared to increase, however variability rendered this result insignificant. Electron spin resonance (ESR) spin trapping, using a-phenyl-N-tert-butylnitrone (PBN) produced a free radical signal from the anoxic-anoxia hyperoxia treated animals which suggests the decrease in MRI T2-weighted image signal intensity was due to free radicals. In Model II, we examined the effects of prolonged normobaric hyperoxia (85% O2) on blood-brain barrier (BBB) integrity and brain phosphorous metabolism. BBB permeability increased following 1 week of hyperoxia. In addition, measurement of high energy phosphates, using in vivo 31P-NMR, showed the PCr/ATP ratio significantly decreased, the ATP/Pi ratio increased and the (ATP+PCr)/Pi ratio increased. Because the BBB is sensitive to oxidative stress its loss of integrity may be due to free radicals. The level of oxidative stress may result in brain elevation of ATP as an adaptation mechanism. In conclusion, anoxic-anoxia and prolonged hyperoxia exposure produce MRI visible changes in the brain. These two mechanisms may be important in the etiology of brain damage observed in many premature infants.

Selective elevation of glutathione levels in target tissues with L-2-oxothiazolidine-4-carboxylate (OTC) protects against hyperoxia-induced lung damage in protein-energy malnourished rats: implications for a new treatment strategy.

It has become recognized that enhancing the antioxidant defense system during the early phase of rehabilitation is important to the survival of wasting protein-energy malnourished (PEM) patients. In this study, we compared the efficacy of dietary protein replenishment and supplementation with L-2-oxothiazolidine-4-carboxylate (OTC, 3.5 mg/d), a cysteine precursor, to protect against hyperoxia-induced lung damage in PEM rats. The PEM rats were produced by feeding weanling rats a protein-deficient diet (0.5% protein) for 14 d. PEM rats were then divided in three dietary treatment groups, 0.5% protein (-Pr), 0.5% protein plus the OTC supplement (+OTC), or 15% protein (+Pr) during 4 d of either hyperoxia (85% O2) or air exposure. Increased lung-to-body weight ratios, indicative of oxidative tissue damage, were observed following exposure to hyperoxia in -Pr and +Pr rats, but not in +OTC rats, even though the OTC supplement and the 15% protein diet contained a comparable amount of cysteine. Tissue reduced glutathione (GSH) status, GSH-dependent enzyme activity and antioxidant defense enzyme activities were monitored in the lung, liver and blood during 4 d of hyperoxia exposure. OTC supplementation enhanced GSH levels significantly in the lung of PEM rats, whereas protein repletion significantly elevated blood GSH concentrations. The protective effect of OTC was not a function of changes in activity of GSH-dependent enzymes or oxygen defense enzymes in the lung. These results indicate that a short-term strategy that selectively elevates GSH levels in the lung is more effective than protein repletion in protecting against hyperoxia-induced oxidative lung damage in PEM rats.

Thiamine supplementation to cancer patients: a double edged sword.

The objectives of this review are to (a) explain the mechanism by which thiamine (vitamin B1) promotes nucleic acid ribose synthesis and tumor cell proliferation via the nonoxidative transketolase (TK) pathway; (b) estimate the thiamine intake of cancer patients and (c) provide background information and to develop guidelines for alternative treatments with antithiamine transketolase inhibitors in the clinical setting. Clinical and experimental data demonstrate increased thiamine utilization of human tumors and its interference with experimental chemotherapy. Analysis of RNA ribose indicates that glucose carbons contribute to over 90% of ribose synthesis in cultured cervix und pancreatic carcinoma cells and that ribose is synthesized primarily through the thiamine dependent TK pathway (> 70%). Antithiamine compounds significantly inhibit nucleic acid synthesis and tumor cell proliferation in vitro and in vivo in several tumor models. The medical literature reveals little information regarding the role of the thiamine dependent TK reaction in tumor cell ribose production which is a central process in de novo nucleic acid synthesis and the salvage pathways for purines. Consequently, current thiamine administration protocols oversupply thiamine by 200% to 20,000% of the recommended dietary allowance, because it is considered harmless and needed by cancer patients. The thiamine dependent TK pathway is the central avenue which supplies ribose phosphate for nucleic acids in tumors and excessive thiamine supplementation maybe responsible for failed therapeutic attempts to terminate cancer cell proliferation. Limited administration of thiamine and concomitant treatment with transketolase inhibitors is a more rational approach to treat cancer.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity.

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 microM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 microM AM. Based on a viability index ((viability of AM-treated cells/viability of controls) x 100%), the Clara cell fraction was significantly (p<0.05) more susceptible than all of the other cell types to 50 microM AM. However, AM cytotoxicity was greatest (p<0.05) in alveolar macrophages following incubation with 100 or 200 microM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 microM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 microM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.