Cultured rat vascular smooth muscle cells are resistant to methylamine toxicity: no correlation to semicarbazide-sensitive amine oxidase.

Methylamine (MA), a component of serum and a metabolite of nicotine and certain insecticides and herbicides, is metabolized by semicarbazide-sensitive amine oxidase (SSAO). MA is toxic to cultured human umbilical vein and calf pulmonary artery endothelial cells. Endothelial cells, which do not exhibit endogenous SSAO activity, are exposed to SSAO circulating in serum. In contrast, vascular smooth muscle cells (VSMC) do exhibit innate SSAO activity both in vivo and in vitro. This property, together with the critical localization of VSMC within the arterial wall, led us to investigate the potential toxicity of MA to VSMC. Cultured rat VSMC were treated with MA (10-5 to 1 M). In some cultures, SSAO was selectively inhibited with semicarbazide or MDL-72145 [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine]. Cytotoxicity was measured via MTT, vital dye exclusion, and clonogenic assays. MA proved to be toxic to VSMC only at relatively high concentrations (LC(50) of 0.1 M). The inhibition of SSAO with semicarbazide or MDL-72145 did not increase MA toxicity, suggesting that the production of formaldehyde via tissue-bound, SSAO-mediated MA metabolism does not play a role in the minimal toxicity observed in isolated rat VSMC. The omission of fetal calf serum (FCS), which contains high SSAO activity, from media similarly showed little effect on cytotoxicity. We conclude that VSMC--in contrast to previous results in endothelial cells--are relatively resistant to MA toxicity, and SSAO does not play a role in VSMC injury by MA.

Developmental vasculotoxicity associated with inhibition of semicarbazide-sensitive amine oxidase.

The endogenous substrate(s) and physiological function(s) of semicarbazide-sensitive amine oxidase (SSAO), a group of enzymes exhibiting highest activity in vascular smooth muscle cells of the mammalian aortic wall, remain undetermined. This study examines the pathophysiological effects in the thoracic aortic wall resulting from specific in vivo SSAO inhibition. Weanling Sprague-Dawley rats were treated acutely or chronically with either semicarbazide hydrochloride or the allylamine derivatives MDL-72274 or MDL-72145 (Marion Merrell Dow Research Institute, Cincinnati, OH). Treatment with these compounds produced acute (6 and 24 h) and chronic (21 day) lowering of SSAO activity in aorta and lung with little effect on the activity of the vital matrix-forming enzyme, lysyl oxidase, in aortas of chronically treated animals. Chronic SSAO inhibition produced lesions consisting of striking disorganization of elastin architecture within the aortic media accompanied by degenerative medial changes and metaplastic changes in vascular smooth muscle cells. No significant difference in the total weight of dry, lipid-extracted aortic elastin and collagen components were observed between chronically SSAO inhibited and control animals. However, the amount of mature elastin was lowered and mature collagen was raised in the aortas of animals treated chronically with semicarbazide. Descending thoracic aortic rings isolated from chronically SSAO-inhibited animals had larger cross-sectional diameters (i.e., exhibited dilation) when compared to corresponding rings from control animals. This study demonstrates that developmental toxicity, characterized by striking vascular lesions and dilated thoracic aortas, can result from specific in vivo SSAO inhibition, suggesting a role for SSAO in connective tissue matrix development and maintenance, and specifically in the development of normal elastin.

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.

Oleander toxicity: an examination of human and animal toxic exposures.

The oleander is an attractive and hardy shrub that thrives in tropical and subtropical regions. The common pink oleander, Nerium oleander, and the yellow oleander, Thevetia peruviana, are the principle oleander representatives of the family Apocynaceae. Oleanders contain within their tissues cardenolides that are capable of exerting positive inotropic effects on the hearts of animals and humans. The cardiotonic properties of oleanders have been exploited therapeutically and as an instrument of suicide since antiquity. The basis for the physiological action of the oleander cardenolides is similar to that of the classic digitalis glycosides, i.e. inhibition of plasmalemma Na+,K+ ATPase. Differences in toxicity and extracardiac effects exist between the oleander and digitalis cardenolides, however. Toxic exposures of humans and wildlife to oleander cardenolides occur with regularity throughout geographic regions where these plants grow. The human mortality associated with oleander ingestion is generally very low, even in cases of intentional consumption (suicide attempts). Experimental animal models have been successfully utilized to evaluate various treatment protocols designed to manage toxic oleander exposures. The data reviewed here indicate that small children and domestic livestock are at increased risk of oleander poisoning. Both experimental and established therapeutic measures involved in detoxification are discussed.

NO2 reactive absorption substrates in rat pulmonary surface lining fluids.

Inhaled 'NO2 is absorbed by a free radical-dependent reaction mechanism that localizes the initial oxidative events to the extracellular space of the pulmonary surface lining layer (SLL). Because 'NO2 per se is eliminated upon absorption, most likely the SLL-derived reaction products are critical to the genesis of 'NO2-induced lung injury. We utilized analysis of the rate of 'NO2 disappearance from the gas phase to determine the preferential absorption substrates within rat SLL. SLL was obtained via bronchoalveolar lavage and was used either as the cell-free composite or after constituent manipulation [(i) dialysis, treatment with (ii) N-ethylmaleimide, (iii) ascorbate oxidase, (iv) uricase, or (v) combined ii + iii]. Specific SLL constituents were studied in pure chemical systems. Exposures were conducted under conditions where 'NO2 is the limiting reagent and disappears with first-order kinetics ([NO2]0 < or = 10 ppm). Reduced glutathione and ascorbate were the principle rat SLL absorption substrates. Nonsulfhydryl amino acids and dipalmitoyl phosphatidylcholine exhibited negligible absorption activity. Whereas uric acid and vitamins A and E displayed rapid absorption kinetics, their low SLL concentrations preclude appreciable direct interaction. Unsaturated fatty acids may account for < or = 20% of absorption. The results suggest that water soluble, low molecular weight antioxidants are the preferential substrates driving 'NO2 absorption. Consequently, their free radicals, produced as a consequence of 'NO2 exposure, may participate in initiating the 'NO2-induced cascade, which results in epithelial injury.

Ozone-reactive absorption by pulmonary epithelial lining fluid constituents.

Previous studies have suggested that the rate of inhaled O3 absorption from the intrapulmonary gas phase is principally mediated by reaction-dependent mechanisms rather than by physical solubility, tissue diffusion, or blood flow (Postlethwait et al., 1994, Toxicol. Appl. Pharmacol. 125, 77-89). The initial site of interaction between O3 and the lung surface occurs at the gas/liquid interface of the epithelial lining fluid (ELF). Therefore, we investigated (a) whether reactive uptake by ELF constituents could account for pulmonary uptake and (b) whether selected constituents acted as O3-specific absorption targets. Rat ELF was harvested by bronchoalveolar lavage. By injecting the same lavage fluid a second [(BALF)2] and/or third [(BALF)3] time into fresh lungs, a more concentrated form of ELF was obtained. Controlled quasi-steady-state exposures (O3 in air; 30-min duration) of cell-free BALF and model substrates (reduced glutathione, GSH) were utilized. Results were based on temperature-specific fractional and normalized uptake rates (r). We observed the following: (1) Buffer pH substantially influenced O3 absorption by GSH but by BALF only modestly. (2) Uptake displayed significant [BALF] and [GSH] dependence. (3) Fractional uptake decreased (BALF and GSH) with increasing [O3] although absolute uptake increased. (4) Absorption demonstrated temperature dependence. Arrhenius plots [ln(r) vs 1/T] were used to compute activation energies (Ea) and Q10. (BALF)1 Ea = 3387 cal/g mol with Q10 = 1.20. GSH (1 mM) Ea = 2240 with Q10 = 1.13. (5) Increasing flow reduced fractional uptake in a nonlinear fashion. (6) Dialysis (1000-molecular-weight cutoff) reduced uptake by (BALF)1 moderately (-30%). Sulfhydryl depletion produced minimal effect (-10%), while ascorbate depletion (-37%) and combined sulfhydryl and ascorbate depletion (-39%) were the most effective. Treatments produced lesser effects on (BALF)3. We conclude that the pH, aqueous substrate, and temperature-dependence and the Ea and Q10 are consistent with reaction-dependent O3 uptake by ELF. The analogous absorption characteristic between the ELF and intact lung (temperature, [O3], contact time) suggests that the ELF represents the primary site for O3-reactive absorption. Reduced sulfhydryls do not appear to substantially interact with inhaled O3. Principal absorption targets may include ascorbate, phospholipids, and other moderate to large molecular weight constituents.

Determinants of inhaled ozone absorption in isolated rat lungs.

Using an isolated rat lung model, we investigated the characteristics of pulmonary O3 absorption, including the contributory role of chemical reaction vs physical solubility. Due to the physicochemical similarities between O3 and NO2, we utilized investigational strategies analogous to those previously employed to characterize NO2 absorption kinetics. The effects of vascular perfusion, temperature, inspired concentration ([O3]i), surface area, and minute ventilation (tidal volume (Vt) times ventilation frequency (f)) on air space O3 clearance during quasi-steady-state exposures were investigated using fractional uptakes (%U) and reactive uptake coefficients (k') as endpoints. We found the following: (1) At 1 ppm [O3]i (37 degrees C), %U (95 +/- 5%) was perfusion independent (60 min). (2) %U displayed temperature dependence (r = 0.99). Activation energies (Ea) and Q10 were computed from Arrhenius plots (ln k' vs 1/T; r = -0.99). For 1 ppm (11-37 degrees C), Ea = 4140 kcal/g.mol and Q10 = 1.23. (3) Absorption demonstrated [O3]i dependence. At 25 degrees C, < or = 1 ppm displayed %U = 86 +/- 4% with k' = 234 ml/min. Exposures > 1 ppm resulted in decreasing %U and k' (5 ppm %U = 60 +/- 3% and k' = 121 ml/min). (4) To evaluate epithelial damage, lactate dehydrogenase (LDH) activity was quantified in cell-free bronchoalveolar lavage fluid. For exposures < or = 1 ppm LDH equaled control, while for exposures > 1 ppm LDH steadily increased to a four-fold maximum at 5 ppm. (5) O3 uptake was independent of functional residual capacity-induced changes in air space surface area. (6) Absorption was proportional to Vt (r = 0.99) and displayed notable ventilation frequency-dependent decline above 70 breaths per minute. Based on the perfusion independence, temperature dependence, and the Ea and Q10, we conclude that O3 absorption in isolated lungs involves a reactive component. While k' remained stable from 0 to 1 ppm O3, at concentrations above 1 ppm other contributory factors such as O3/substrate reaction kinetics, epithelial damage, and solute O3 backpressure may affect the overall net absorption rate. In addition, the data suggest that O3 uptake may be principally localized to the conducting airways.

Kinetics of NO2 air space absorption in isolated rat lungs.

We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO2 interfacial transfer into ELF exhibits first-order kinetics with respect to NO2, attains [NO2]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510, 1991). We have extended these observations by evaluating the kinetics of NO2 gas phase disappearance in isolated ventilating rat lungs. Transient exposures (2-3/lung at 25 degrees C) employed rebreathing (NO2-air) from a non-compliant continuously stirred closed chamber. We observed that 1) NO2 uptake rate is independent of exposure period, 2) NO2 gas phase disappearance exhibited first-order kinetics [initial rate (r*) saturation occurred when [NO2] > 11 ppm], 3) the mean effective rate constant (k*) for NO2 gas phase disappearance ([NO2] < or = 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 +/- 5 ml/min, 4) with [NO2] < or = 11 ppm, k* and r* were proportional to tidal volume, and 5) NO2 fractional uptakes were constant across [NO2] (< or = 11 ppm) and tidal volumes but exceeded quasi-steady-state observations. Preliminary data indicate that this divergence may be related to the inspired PCO2. These results suggest that NO2 reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF.(ABSTRACT TRUNCATED AT 250 WORDS)

Interfacial transfer kinetics of NO2 into pulmonary epithelial lining fluid.

Previous studies, both in intact lungs and epithelial lining fluid (ELF) (J. Appl. Physiol. 68: 594-603, 1990 and J. Appl: Physiol. 69: 523-531, 1990), have suggested that the steady-state absorption of inhaled NO2 is mediated by chemical reaction(s) between NO2 and ELF solute reactants. To characterize the kinetics of NO2 absorption into aqueous biological substrates across a gas-liquid interface, we utilized a closed system of known geometry and initial gas phase [NO2] [([NO2]g)0] to expose ELF (as bronchoalveolar lavage; BAL) and a biochemical model system (glutathione, GSH). Assessments of NO2 reactive uptake, into both GSH and ELF, indicated first-order NO2 kinetics [([NO2]g)0 less than or equal to 10.5 ppm] with effective rate constants of (kNO2)GSH = 4.8 and (kNO2)BAL = 2.9 ml.min-1.cm-2 (stirred). Above 10.5 ppm (1 mM GSH), zero-order kinetics were observed. Both (kNO2)GSH and (kNO2)BAL showed aqueous reactant dependence. The reaction order with respect to GSH and BAL was 0.47 and 0.64, respectively. We found no effect of interfacial surface area or bulk phase volume on kNO2. In unstirred systems, significant interfacial resistance was observed and was related to reactant concentration. These results indicate that NO2 reactive uptake follows first-order kinetics with respect to NO2 ([NO2]g less than or equal to 10.5 ppm) and displays aqueous substrate dependence. Furthermore the site of reactive absorption appears to be limited to near the aqueous surface interface. Unstirred conditions confine interfacial mass transfer kinetics in a dose-dependent manner. These phenomenological coefficients may provide the basis for direct extrapolation to environmentally relevant exposure concentrations.

Transfer of NO2 through pulmonary epithelial lining fluid.

Absorption of inhaled NO2 across the pulmonary gas/tissue interface is principally governed by chemical reaction(s) rather than by physical solubility. While the kinetics of NO2 transfer into reactant-containing aqueous solutions appear to be bulk phase independent, it is unclear whether unreacted NO2 diffuses appreciably through the epithelial lining fluid (ELF) to cellular compartments. We avoided the difficulties associated with directly quantifying NO2 dissolved in biological fluids by indirectly determining the potential for NO2 penetration to underlying tissues. An in vitro system was developed which horizontally suspended a wettable, gas permeable, fibrous material between two gas chambers. Aqueous substrates were applied to the sieve material and NO2 (10.9 ppm) was introduced into one chamber and sampled for in the other. O2 served as a tracer gas. We determined the influence of ELF, a model biochemical (reduced glutathione; GSH), and PO4 buffer (control) on NO2 transfer as evaluated by "breakthrough time." (A) Both O2 and NO2 rapidly diffused through the sieve material when dry. Under PO4 wetted conditions, O2 continued to penetrate rapidly but NO2 transfer was slightly inhibited relative to O2. (B) Addition of GSH (1 mM) significantly prolonged NO2 breakthrough time. Increasing initial [GSH] resulted in concomitant prolongation of NO2 breakthrough time. (C) We observed a direct correlation between oxidation of sieve GSH and NO2 breakthrough. (D) Freshly harvested rat ELF inhibited NO2 transfer in a concentration-dependent manner similar to GSH. These data suggest that in the presence of reactant solutes, unreacted NO2 does not penetrate through the ELF layer. Reactive absorption must, therefore, occur primarily within the ELF compartment so that reaction products which induce subsequent toxicity are generated as a result of the initial uptake interactions.

Reactive absorption of nitrogen dioxide by pulmonary epithelial lining fluid.

In a previous study (J. Appl. Physiol. 68: 594-603, 1990) in isolated rat lungs, we suggested that the rate of pulmonary air space absorption of inhaled NO2 is limited, in part, by chemical reaction(s) rather than by physical solubility. Because the initial site of primary absorption interactions involves the epithelial lining fluid (ELF), we investigated whether ELF-NO2 interactions could account for pulmonary NO2 reactive absorption. Rat ELF, obtained by bronchoalveolar lavage (BAL), was compared with a model chemical system (reduced glutathione, GSH). In vitro exposures (NO2-air) used constant gas flow and planar gas-liquid interfaces. 1) Solvent pH notably altered NO2 uptake by GSH but to a lesser extent by BAL. 2) Uptake displayed [GSH]-dependent saturation. [ELF] in BAL was augmented by sequential lavage (lavagate reuse) of multiple lungs. Uptake was proportional to [ELF] but did not saturate under these exposure conditions. 3) The uptake rate exhibited [NO2] dependence. However, relative to increasing [NO2], fractional uptakes decreased for BAL and 1 mM GSH but not for 10 mM GSH. 4) Altered convective gas flow produced nonlinear increments in uptake (10 mM GSH) and substantial decrements in fractional uptake. 5) Arrhenius plots [ln(r) vs. 1/T, where r is reaction rate and T is absolute temperature (degree K)] for BAL and 1 mM GSH yielded respective activation energies of 4,952 and 4,149 kcal.g-1.mol-1 and degree of change in the rate of NO2 uptake per 10 degrees C (Q10) of 1.32 and 1.25. These results imply that the rate of NO2 uptake into rat ELF, like intact lung, is limited, in part, by chemical reaction(s).(ABSTRACT TRUNCATED AT 250 WORDS)