Survival on biventricular mechanical support with the Centrimag® as a bridge to decision: a single-center risk stratification.

OBJECTIVE: Temporary mechanical assist devices are increasingly being used as a lifesaving bridge to decision in patients requiring cardiopulmonary resuscitation. We report our single-center experience with biventricular Centrimag® pumps over a five-year period. METHOD: Data was retrospectively collected in consecutive patients who required biventricular support from 2008 to 2013. Patients who were supported with central cannulation using the Centrimag® system were analyzed. In addition to demographic information, data pertaining to indications, outcomes and mortality were collected. RESULTS: The cohort consisted of 48 patients (19 women and 29 men, mean age of 56 years). The median duration of support was 14 days. The median duration to patient expiration while still on the Centrimag® was 12 days. Thirty-day survival was 56% (27/48). Nine patients were explanted to recovery, while fourteen patients were converted to a durable LVAD, two of whom were then transplanted. We stratified patients into two groups. Group I comprised patients who were either explanted to recovery, converted to durable LVAD or transplanted (23/48) and Group II consisted of patients who either died on the Centrimag® or were explanted for withdrawal of care (25/48). Statistical analysis did not reveal any clinically significant differences between the two groups in terms of age, sex, etiology, hemodynamic, co-morbidities or laboratory parameters. CONCLUSION: The biventricular Centrimag® can be used as a bridge to decision in patients with thirty-day survival of >50%. Parameters to predict 30-day survival in this high-risk cohort continue to remain elusive.

Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2 knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors.

Styrene (S) is lung tumorigenic in mice but not in rats. S and its alkene-oxidized metabolite styrene oxide (SO) were not lung toxic in CYP2F2(-/-) [knockout] mice, indicating S-induced mouse lung tumors are mediated through mouse-specific CYP2F2-generated ring-oxidized metabolite(s) in lung bronchioles. The human relevance of the CYP2F MOA was assessed by insertion of a human CYP2F1, 2A13, 2B6 transgene into CYP2F2(-/-) mice; CYP2F1 expression and activity were confirmed in the transgenic (TG) mice. No evidence of cytotoxicity or increased cell proliferation (BrdU labeling) was seen in TG mice treated with either S or SO (200mg/kg/day ip for 5days). In contrast to S and SO, 4HS (105mg/kg/day ip for 5days) increased BrdU labeling 5-10-fold in WT mice, <3-fold increase in KO mice and 2-4-fold in TG mice. The limited response of 4HS in KO and TG mice may result from intrinsic toxicity or from further metabolism; regardless of the MOA, these findings indicate that the CYP2F-mediated tumorigenic MOA in WT mice is not operative for S, SO, or for 4HS putatively derived from metabolism of S by CYP2F1 in humans, and thus S-induced mouse lung tumors are unlikely to be relevant to human risk.

Effect of ammonium sulfate fertilization on bahiagrass quality and copper metabolism in grazing beef cattle.

To assess the impact of S fertilization on bahiagrass (Paspalum notatum) quality and Cu metabolism in cattle, two studies were conducted during the summer grazing season (1999 and 2000). Pasture replicates (16.2 ha; n = 2/treatment) received the same fertilizer treatment in each growing season, consisting of 1) 67 kg N/ha from ammonium sulfate (AS), 2) 67 kg N/ha from ammonium nitrate (AN), and 3) control (no fertilizer; C). Forage sampling was conducted at 28-d intervals following fertilization by the collection of whole plants (four samples/pasture) in randomly distributed 1-m2 grazing exclusion cages and analyzed for CP, in vitro organic matter digestibility, S, P, Ca, K, Mg, Na, Fe, Al, Mn, Cu, and Zn. To determine the effect of fertilizer treatment on liver trace mineral concentrations in grazing cattle, random liver tissue samples were collected (n = 12; four/treatment) at the start and end of the study period in 2000. Ammonium sulfate fertilization increased (P < 0.001) forage S concentration in both years. Plant tissue N concentrations were increased by N fertilization, regardless of source, in 2000, but not in 1999. Cows grazing AS pastures had lower (P < 0.05) liver Cu concentrations at the end of the study period in 2000 compared to AN and C. In Exp. 2, 37 Cu-deficient heifers grazing AS fertilized pastures were obtained from the same location and allocated to one of two treatments, consisting of supplements providing 123 mg/d of either inorganic (Cu sulfate; n = 12) or organic (Availa-Cu; n = 15) Cu. Treatments were delivered for 83 d. Liver Cu increased over time in all heifers regardless of treatment; however, heifers supplemented with Availa-Cu tended (P = 0.09) to have higher mean liver Cu concentrations than those receiving Cu sulfate. The results of these studies indicate that AS fertilization of bahiagrass increases forage S concentrations. When provided free-choice access to a complete salt-based trace mineral supplement, cows grazing AS-fertilized pastures had lower liver Cu concentrations than cows grazing pastures fertilized with AN; upon removal from high-S pastures, cattle were able to respond to Cu supplementation.

Effect of copper source and level on the rate and extent of copper repletion in Holstein heifers.

The objective of this study was to evaluate the rate and extent of Cu repletion in Holstein heifers using two Cu sources (organic and inorganic) at two levels (15 and 30 mg/kg). An additional repletion treatment included a Cu oxide bolus. Heifers (n = 50) were individually fed a total mixed ration fortified with S and Mo at 0.40%, and 15 mg/kg of dry matter of the total diet, respectively. After 111 d of depletion, heifers were stratified by liver Cu concentration and randomly allotted to one of five repletion treatments. Four treatments consisted of feed sources of Cu (feed-Cu), 1) CuSO4 at 15 mg/kg; 2) CuSO4 at 30 mg/kg; 3) Availa-Cu at 15 mg/kg; and 4) Availa-Cu at 30 mg/kg. Availa-Cu is an organic Cu source that produces a Cu-amino acid complex. A fifth treatment, consisting of an intraruminal bolus (IB), provided a single dose of 25 g of CuO needles. Repletion treatments were delivered in the same total mixed ration without supplemental S and Mo. Copper status was assessed in blood and liver samples collected on 14-d intervals for 70 d. Irrespective of treatment, all heifers increased in body weight during the repletion period. Liver Cu increased in each feed-Cu treatment over time. Heifers treated with an IB reached a peak in liver Cu concentration (165.5 mg/kg) on d 28. Mean liver Cu concentrations were higher in heifers receiving 30 mg/kg of Cu compared with heifers receiving 15 mg/kg of Cu. Red blood cell superoxide dismutase (SOD) activity was higher (P < 0.001) in heifers receiving CuSO4 than Availa-Cu (0.98 vs 0.87 U). Also, SOD activity was higher when heifers were supplemented with 30 vs 15 mg/kg Cu (0.98 vs 0.87 U). Heifers receiving the Cu IB had higher SOD activity than heifers receiving feed-Cu sources (1.03 vs 0.92 U). Plasma ceruloplasmin concentration was higher (P < 0.001) in IB-treated heifers vs. other treatments. No differences in plasma ceruloplasmin were detected for feed-Cu source or level. These results indicate that all Cu sources evaluated in this study elevated Cu status of depleted heifers, particularly when provided at higher dietary levels.

Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle.

OBJECTIVE: To evaluate viral and bacterial respiratory pathogens and Mycoplasma spp isolated from lung tissues of cattle with acute interstitial pneumonia (AIP) and cattle that had died as a result of other causes. SAMPLE POPULATION: 186 samples of lung tissues collected from cattle housed in 14 feedlots in the western United States. PROCEDURE: Lung tissues were collected during routine postmortem examination and submitted for histologic, microbiologic, and toxicologic examinations. Histologic diagnoses were categorized for AIP, bronchopneumonia (BP), control samples (no evidence of disease), and other disorders. RESULTS: Cattle affected with AIP had been in feedlots for a mean of 1272 days before death, which was longer than cattle with BP and control cattle. Detection of a viral respiratory pathogen (eg, bovine respiratory syncytial virus [BRSV], bovine viral diarrhea virus, bovine herpesvirus 1, or parainfluenza virus 3) was not associated with histologic category of lung tissues. Bovine respiratory syncytial virus was detected in 8.3% of AIP samples and 24.0% of control samples. Histologic category was associated with isolation of an aerobic bacterial agent and Mycoplasma spp. Cattle with BP were at greatest risk for isolation of an aerobic bacterial agent and Mycoplasma spp. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results suggests that AIP in feedlot cattle is not a consequence of infection with BRSV. The increased, risk of isolation of an aerobic bacterial agent from cattle with AIP, compared with control cattle, may indicate a causal role or an opportunistic infection that follows development of AIP.

Association of 3-methyleneindolenine, a toxic metabolite of 3-methylindole, with acute interstitial pneumonia in feedlot cattle.

OBJECTIVE: To compare concentrations of 3-methyleneindolenine (3MEIN) in lung tissues obtained from feedlot cattle that died as a result of acute interstitial pneumonia (AIP) and cattle that died as a result of other causes and to compare blood concentrations of 3MEIN in healthy feedlot cattle and feedlot cattle with AIP. STUDY POPULATION: Blood samples and lung tissues collected from 186 cattle housed in 14 feedlots in the western United States. PROCEDURE: Samples of lung tissues were collected during routine postmortem examination and submitted for histologic, microbiologic, and toxicologic examination. Blood samples were collected from cattle with clinical manifestations of AIP and healthy penmates. Histologic diagnoses were categorized as AIP, bronchopneumonia (BP), control samples, and other disorders. Concentrations of 3MEIN were determined in lung tissues and blood samples, using an ELISA. RESULTS: Concentrations of 3MEIN in lung tissues were significantly greater in AIP and BP samples, compared with control samples. Absorbance per microgram of protein did not differ between BP and AIP samples. Blood concentrations of 3MEIN were significantly greater in cattle with AIP, compared with healthy cattle or cattle with BP. Odds of an animal with AIP being a heifer was 3.1 times greater than the odds of that animal being a steer. CONCLUSIONS AND CLINICAL RELEVANCE: Increased pulmonary production of 3MEIN may be an important etiologic factor in feedlot-associated AIP.

Detection and characterization of DNA adducts of 3-methylindole.

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

Selective dehydrogenation/oxygenation of 3-methylindole by cytochrome p450 enzymes.

3-Methylindole (3 MI) is a selective pulmonary toxicant, and cytochrome P450 (P450) bioactivation of 3 MI, through hydroxylation, epoxidation, or dehydrogenation pathways, is a prerequisite for toxicity. CYP2F1 and CYP2F3 exclusively catalyze the dehydrogenation of 3 MI to 3-methyleneindolenine, without detectable formation of the hydroxylation or epoxidation products. It was not known whether 3 MI is simply an excellent dehydrogenation substrate for all P450 enzymes, or whether certain cytochrome P450s responsible for 3 MI bioactivation have unique active sites that only catalyze the dehydrogenation of the molecule, while other P450s would catalyze only the oxygenation of 3 MI. Therefore, the kinetics of product formation by the CYP2F1 and CYP2F3 enzymes were compared with other cytochrome P450 enzymes. The enzymes tested were CYP1A1, CYP1A2, CYP1B1, and CYP2E1. The CYP1A1 and CYP1A2 enzymes produced all three 3 MI metabolites: the dehydrogenation product, 3-methyleneindolenine (V(max)/K(m) = 4 and 22, respectively); the hydroxylation product, indole-3-carbinol (V(max)/K(m) = 42 and 100, respectively); and the epoxidation product, 3-methyloxindole (V(max)/K(m) = 4 and 72, respectively). These CYP1A enzymes catalyzed oxygenation of 3 MI at much faster rates than dehydrogenation. CYP1B1 produced indole-3-carbinol (V(max)/K(m) = 85) and 3-methyloxindole (V(max)/K(m) = 7), and CYP2E1 only produced 3-methyloxindole (V(max)/K(m) = 98), but neither enzyme catalyzed the formation of the dehydrogenated product. Six additional P450 enzymes that were tested formed none of the dehydrogenation product. The ability of the various CYP1 family enzymes to catalyze the formation of all three major 3 MI metabolites, along with the specific oxygenation by CYP2E1, illustrates that dehydrogenation of 3 MI is not a substrate-directed process, but that the members of the CYP2F family possess unique active sites that specifically catalyze only the dehydrogenation mechanism.

Determination of capsaicin, dihydrocapsaicin, and nonivamide in self-defense weapons by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

Sensitive and selective liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the analysis of capsaicin, dihydrocapsaicin, and nonivamide in pepper spray products have been developed. Chromatographic separation of the capsaicinoid analogues was achieved using a reversed-phase HPLC column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantification of the capsaicinoids was achieved by electrospray ionization single-stage mass spectrometry monitoring the protonated molecules of the internal standard (m/z 280), capsaicin (m/z 306), dihydrocapsaicin (m/z 308), and nonivamide (m/z 294) or by tandem mass spectrometry monitoring the appropriate precursor-to-product-ion transitions. The plot of concentration versus peak area ratio was linear over the range of 10-750 ng/ml using LC-MS and 10-500 ng/ml using LC-MS-MS. However, to accurately quantify the capsaicinoids in the pepper spray products calibration curves between 10 and 1000 ng were constructed and fit using a weighted quadratic equation. Using the quadratic curve, the accuracy of the assay ranged from 91 to 102% for all analytes. The intra-assay precision (RSD) for capsaicin was 2% at 25 ng/ml, 10% at 500 ng/ml, and 3% at 800 ng/ml. The inter-assay precision (RSD) for capsaicin was 6% at 25 ng/ml, 6% at 500 ng/ml, and 9% at 800 ng/ml. Similar values for inter- and intra-assay precision were experimentally obtained for both dihydrocapsaicin and nonivamide. The analysis of selected pepper spray products demonstrated that the capsaicinoid concentration in the products ranged from 0.7 to 40.5 microg/microl.

Quantitative analysis of capsaicinoids in fresh peppers, oleoresin capsicum and pepper spray products.

Liquid chromatography-mass spectrometry was used to identify and quantify the predominant capsaicinoid analogues in extracts of fresh peppers, in oleoresin capsicum, and pepper sprays. The concentration of capsaicinoids in fresh peppers was variable. Variability was dependent upon the relative pungency of the pepper type and geographical origin of the pepper. Nonivamide was conclusively identified in the extracts of fresh peppers, despite numerous reports that nonivamide was not a natural product. In the oleoresin capsicum samples, the pungency was proportional to the total concentration of capsaicinoids and was related by a factor of approximately 15,000 Scoville Heat Units (SHU)/microg of total capsaicinoids. The principle analogues detected in oleoresin capsicum were capsaicin and dihydrocapsaicin and appeared to be the analogues primarily responsible for the pungency of the sample. The analysis of selected samples of commercially available pepper spray products also demonstrated variability in the capsaicinoid concentrations. Variability was observed among products obtained from different manufacturers as well as from different product lots from the same manufacturer. These data indicate that commercial pepper products are not standardized for capsaicinoid content even though they are classified by SHU. Variability in the capsaicinoid concentrations in oleoresin capsicum-based self-defense weapons could alter potency and ultimately jeopardize the safety and health of users and assailants.

Significance of grunting respirations in infants admitted to a well-baby nursery.

OBJECTIVES: (1) To determine the frequency and duration of grunting in term and near-term newborns; (2) to determine the peripartum characteristics associated with grunting; and (3) to compare the short-term outcomes of newborns with and without grunting. DESIGN: Medical record review of all newborns admitted to a well-baby nursery during a 2-month period. SETTING: University well-baby nursery for term infants, with more than 2700 deliveries annually. MAIN OUTCOME MEASURES: Frequency and duration of grunting, maternal and newborn clinical characteristics, clinical course, and length of stay. RESULTS: Grunting respirations beginning during the first 4 hours of life were recorded for 81 (17.4%) of 466 newborns. Fifty-five (68%) stopped grunting within 30 minutes of birth, 69 (85%) by 1 hour, and 75 (93%) by 2 hours. More mothers of grunting infants received intrapartum antibiotics than mothers of nongrunters (33% vs 20%; P =.03). More grunting infants than nongrunters received bag and mask resuscitation (15% vs 5%; P =.01). More chest radiographs, blood cell counts, and blood cultures were ordered for grunting infants, and antibiotics were more often given to grunting than nongrunting infants (11.1% vs 4.6%; P =.04). Grunters' length of stay exceeded that of nongrunters (72 vs 55 hours; P =.01), but only 3 were transferred to a neonatal intensive care unit. CONCLUSIONS: All grunting infants should be carefully observed, but because nearly all otherwise healthy term or near-term infants will stop grunting and have a benign course, other interventions can be postponed for 1 or 2 hours to give the newborn a chance to stop grunting or show other signs of respiratory illness.

Characterization of acute interstitial pneumonia in cattle in southern Alberta feedyards.

Field data were collected over 2 consecutive years to characterize acute interstitial pneumonia (AIP) in feedyard cattle. Thirty-eight cattle with clinical symptoms of AIP were examined following emergency slaughter; 31 (all heifers) were confirmed to have AIP on the basis of gross and histological lung pathology. The 7 without AIP, plus 17 asymptomatic penmates, were used as contemporary controls. Plasma concentrations of 3-methylindole (3MI) metabolites were higher (P < 0.001) in heifers afflicted with AIP than in the control animals, and concentrations of 3MI mercapturates in the urine were lower (P < 0.007) in affected heifers. Concentrations of 3MI adducts in lung tissue and in microsomal protein did not differ (P > 0.05) between the 2 groups, and 3MI was not detected in ruminal fluid from either group. Total ruminal bacterial numbers and populations of lactobacilli and protozoa were similar (P > 0.05) between the AIP-positive and unafflicted groups, but fewer (P < 0.05) cellulolytic bacteria were present in the positive group. Bovine respiratory syncytial virus antigen was not found in lung tissue from any of the heifers confirmed to have AIP. To our knowledge, this study is the first to implicate 3MI metabolites as having a role in feedyard AIP. Further research is required to determine the factors responsible for the elevation in 3MI adducts in plasma and urine of feedyard cattle afflicted with AIP.

Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes.

A study was conducted to investigate qualitative and quantitative aspects of the phase I metabolism of 3-methylindole (3MI) by porcine liver microsomes. Microsomal suspensions were prepared from the liver of 30 intact (uncastrated) male pigs. Metabolites produced in microsomal incubations were identified and quantitated with HPLC-UV, HPLC-fluorescence, and UV-spectral analysis; liquid chromatography-mass spectrometry (LC-MS) and NMR were used for the identification of a metabolite for which a reference compound was not available. The results showed that seven major metabolites of 3MI are produced by porcine microsomes, three of which had already been identified in pigs (3-OH-3-methyloxindole, 5-OH-3-methylindole, and 6-OH-3-methylindole). The other four major 3MI metabolites identified were 3-OH-3-methylindolenine, 3-methyloxindole, indole-3-carbinol, and 2-aminoacetophenone. On average, the metabolite that was produced in larger amounts was 3-OH-3-methylindolenine (45.1%), followed by the two oxindoles 3-methyloxindole (27.9%) and 3-OH-3-methyloxindole (18.5%). Average percentage of production of 6-OH-3-methylindole was 4.9%, whereas indole-3-carbinol accounted for 2.7% of all metabolites produced; 2-amino-acetophenone and 5-OH-3-methylindole were the metabolites produced in lesser amounts (0.5 and 0.3%, respectively). Large interindividual differences in the rate of production of all metabolites were observed. This variation could be attributed to differences in the activity and/or level of expression of phase I biotransformation enzymes and this issue should be further investigated.

Specific dehydrogenation of 3-methylindole and epoxidation of naphthalene by recombinant human CYP2F1 expressed in lymphoblastoid cells.

3-Methylindole (3MI) is a naturally occurring pulmonary toxin that requires metabolic activation. Previous studies have shown that 3MI-induced pneumotoxicity resulted from cytochrome P-450-catalyzed dehydrogenation of 3MI to an electrophilic methylene imine (3-methyleneindolenine), which covalently bound to cellular macromolecules. Multiple cytochrome P-450s are capable of metabolizing 3MI to several different metabolites, including oxygenated products. In the present study, the role of human CYP2F1 in the metabolism of 3MI was examined to determine whether it catalyzes dehydrogenation rather than hydroxylation or ring oxidation. Metabolism was examined using microsomal fractions from human lymphoblastoid cells that expressed the recombinant human CYP2F1 P-450 enzyme. Expression of CYP2F1 in the lymphoblastoid cells proved to be an appropriate expression system for this enzyme. Products were analyzed using HPLC and the mercapturate, 3-[(N-acetylcystein-S-yl)methyl]indole, of the reactive intermediate was identified and quantified. Product analysis showed that human CYP2F1 efficiently catalyzed the dehydrogenation of 3MI to the methylene imine without detectable formation of indole-3-carbinol or 3-methyloxindole. High substrate concentrations of 3MI strongly inhibited production of the dehydrogenated product, a result that may indicate the existence of mechanism-based inhibition of CYP2F1 by 3MI. Recombinant CYP2F1 demonstrated remarkable selectivity for the bioactivation of 3MI to the putative dehydrogenated reactive electrophile. Bioactivation of naphthalene to its pneumotoxic epoxide by CYP2F1 was also demonstrated.

Effect of melengestrol acetate on development of 3-methylindole-induced pulmonary edema and emphysema in sheep.

The involvement of melengestrol acetate (MGA) in susceptibility to developing pulmonary edema and emphysema following oral administration of 3-methylindole (3MI) was investigated using 10 Suffolk ewes receiving 0 or 0.15 mg of MGA daily (n = 5). Blood, urine and ruminal fluid were collected immediately prior to 3MI dosing (0.2 g/kg BW) and 1, 2, 3, 4, 5, 6, 12 and 24 h (blood); 3, 6, 9, 12 and 15 h (urine) and 1, 2, 3 and 12 h (ruminal fluid) afterward. Ewes receiving MGA experienced earlier (P < 0.05) onset of respiratory distress than the control ewes (2.5 vs 4 h), and upon euthanasia at 96 h, their lung weight relative to body weight tended (P < 0.10) to be lower. Ruminal 3MI concentrations did not differ between treatments (P > 0.05). Ewes receiving MGA had higher (P < 0.05) concentrations of 3MI metabolites in plasma prior to dosing than did control ewes, and these values tended to remain higher throughout the sampling period. Immunoreactivity assays indicated more pneumotoxin present in the lungs of MGA-treated ewes than controls. Lung damage was apparently more acute and accelerated in the MGA-treated ewes than in the controls. Urinary 3MI mercapturate concentrations differed (control > MGA-treated, P < 0.05) at 9, 12, and 15 h, but this difference was not apparent when urinary production (as estimated by creatinine concentration) was considered. The implications of these findings for MGA-treated feedlot heifers are currently under investigation.

Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole.

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.

Evidence supporting the formation of 2,3-epoxy-3-methylindoline: a reactive intermediate of the pneumotoxin 3-methylindole.

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isotope techniques and mass spectrometry. Determination of hydride shift and incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3-methyloxindole, metabolites that may be in part dependent on the presence of the epoxide, were utilized as indicators of the epoxide's existence. One mechanism for the formation of 3-methyloxindole involves cytochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung microsomes with [2-2H]-3-methylindole, the retention of 2H in 3-methyloxindole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenine is an imine reactive intermediate that could be produced by ring opening of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-methyloxindole by the cytosolic enzyme aldehyde oxidase. Activities of this putative detoxification enzyme were determined in both hepatic and pulmonary tissues from goats, rats, mice, and rabbits, but the activities could not be correlated to the relative susceptibilities of the four species to 3-methylindole toxicity. The 18O incorporation into either 3-methyloxindole or 3-hydroxy-3-methyloxindole from both 18O2 and H218O was determined. The 18O incorporation into 3-methyloxindole from 18O2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 18O into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence of two new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate.

Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective pneumotoxins 3-methylindole and naphthalene.

Members of the CYP2F gene subfamily are selectively expressed in lung tissues and have been implicated as important catalysts in the formation of reactive intermediates from several pneumotoxic chemicals. Human CYP2F1 bioactivates 3-methylindole (3MI), while mouse CYP2F2 bioactivates naphthalene. Although 3MI is a potent pneumotoxin in ruminants and rodents, the participation of cytochrome P450s from the 2F subfamily in 3MI bioactivation has not been fully defined. To test the hypothesis that a goat lung 2F homologue uniquely catalyzes the dehydrogenation of 3MI to the putative electrophile 3-methylene-indolenine, the CYP2F3 cDNA was cloned from a goat lung cDNA library and expressed in Escherichia coli. The predicted amino acid sequence of CYP2F3 possessed 82% identity to both human CYP2F1 and mouse CYP2F2. CYP2F3 was mutated at the 5' end, expressed in E. coli, and shown to have a molecular mass of 50 kDa. The reconstituted enzyme uniquely catalyzed only the dehydrogenation of 3MI to form 3-methylene-indolenine, an electrophilic intermediate, without detectable formation of other products, thus demonstrating highly unusual selectivity for dehydrogenation rather than hydroxylation of a substrate. Immunoinhibition studies demonstrated that about 20% of the production of the intermediate in goat lung microsomal samples was produced by CYP2F3. The CYP2F3 enzyme had a specific activity that was similar to that of human cDNA-expressed CYP2F1. CYP2F3 also stereoselectively catalyzed the formation of the 1R,2S-oxide from naphthalene; this stereoisomer is the putative pneumotoxin. The enzyme, however, lacked catalytic activity with other common P450 substrates including 7-ethoxycoumarin, a substrate for CYP2F1, indicating that the substrate selectivity of CYP2F3 appears to be high.

Production and characterization of specific antibodies: utilization to predict organ- and species-selective pneumotoxicity of 3-methylindole.

3-Methylindole (3MI) selectively causes damage to pulmonary tissues; the species-selective order is goats, rats, and rabbits, with rabbits sustaining the least damage. 3MI is bioactivated to toxic intermediates by cytochrome P450 enzymes. Covalent binding of the electrophilic 3-methyleneindolenine intermediate to proteins is a likely mechanism of 3MI-mediated lung damage. Polyclonal antibodies were developed to thioether adducts of 3-methyleneindolenine and were shown by competitive enzyme-linked immunosorbent assay (ELISA) to be highly selective for the detection of 3MI adducts. Rabbits, rats, and goats were treated with 350, 400, and 15 mg/kg 3MI, respectively. The lungs, liver, and kidneys of each animal were collected 24 hr later and tissue fractions were analyzed by ELISA. Lung tissue fractions from goat (pellet, cytosol, and microsomes) had greater immunoreactivity than those from rat. Immunoreactivity in rat tissues was greater than that in rabbit tissues. In all of the animals, lung had greater immunoreactivity than kidney, and kidney had greater reactivity than liver. These studies demonstrate that thioether adducts of 3MI with proteins can be detected specifically by these antisera, and the adducts are precisely correlated to species and tissue susceptibility of 3MI. In addition, human lung and liver samples were moderately immunoreactive. Therefore, humans form adducts of 3MI in these tissues and are predicted to be susceptible to 3MI-mediated toxicity.