Roy J. Richards

Depletion of urate in human nasal lavage following in vitro ozone exposure

Ozone, a strong oxidant present in summer smog, is thought to primarily react with antioxidant molecules found in the epithelial lining fluid of the respiratory tract. In humans, as much as 40% of inhaled ozone can be removed in the nasal cavity where the major extracellular antioxidant has been identified as uric acid. The present study was undertaken to examine urate/oxidant interactions in human nasal lavage fluid following in vitro exposure to ozone at concentrations relevant to the U.K. Lavage fluid was collected from 8 volunteers using a modified Foley catheter which permits prolonged contact of isotonic saline with the anterior nasal cavity. Nasal lavage samples in multiwell plates were exposed to ozone at concentrations of 50, 100 and 250 ppb. Samples were removed at intervals from 15 to 240 min following exposure and assayed for uric acid depletion. Uric acid concentrations in the nasal lavage were found to fall from 8.52 (time zero) to 3.99 microM, 0.05 and 0.07 microM after 240 min at 50, 100 and 250 ppb ozone respectively. At a non-environmentally relevant ozone concentration of 1000 ppb, uric acid was completely depleted after 60 min. Regression analysis showed a linear correlation between rate of loss of urate and ozone concentration (R2 = 0.97). A novel, non-invasive technique is described to investigate antioxidant compromise and its importance in individual subjects. We conclude that uric acid in nasal lavage samples is scavenged by ozone in a dose and time dependent manner.

Interactions between paraquat, endogenous lung amines' antioxidants and isolated mouse Clara cells

The ability of paraquat to damage mouse lung Clara cells in the presence and absence of herbicide inhibitors is investigated using a cell culture system. Clara cell damage is assessed on the loss of nitroblue tetrazolium reductase activity and the inability to attach and spread on an extracellular matrix. Endogenous amines such as putrescine and spermidine reduce paraquat-induced damage at low concentrations indicating that they compete for the same cell surface receptor as paraquat and thus potentially block the accumulation of the herbicide. The efficacy of 10 microM exogenous putrescine as a protectant is reduced the longer the time before it is added to the cultures. Clara cells contain high levels of NADPH-dependent P-450 reductase which is required to redox cycle the paraquat and generate reactive oxygen radicals. Compounds with antioxidant properties are examined for their ability to reduce the Clara cell damage. Cystamine, the disulphide form of the naturally occurring thiol, cysteamine, and taurine, a metabolite of cystamine, both of which are accumulated in the lung, do not reduce paraquat-induced Clara cell damage. Another antioxidant, alpha-tocopherol is also ineffective but reduced glutathione (GSH), present in high quantities (3.2 mM) in clara cells, could reduce damage to the cultured cells. Cysteine, a precursor of GSH, can also prevent Clara cell damage when the concentration of paraquat is low.

The accumulation of pentamidine into rat lung slices and its interaction with putrescine

The aromatic diamidine, pentamidine, accumulated into rat lung slices by an uptake system that obeyed saturation kinetics, with an average Km value of 554 microM and a Vmax value of 4077 nmol/g lung wet wt/30 min, respectively. This system was not inhibited by metabolic inhibitors but was greatly diminished by lowering the temperature from 37 degrees to 4 degrees. Both compounds, pentamidine and putrescine, inhibited the uptake of the other and the inhibition of pentamidine accumulation by putrescine was demonstrated to be non-competitive. Uptake of putrescine was inhibited by increasing concentrations of pentamidine. As putrescine accumulates in epithelial type 1 and type 2 cells and in Clara cells, it is likely that pentamidine is also accumulated in these cell types but does not utilize the pulmonary uptake system for polyamine transport. Within the time period studied, toxic effects of the drug were not observed.

The accumulation of pentamidine and the toxic effects of the drug, its selected analogues and metabolites on isolated alveolar cells

Radiolabelled [3H]pentamidine is accumulated into 48-h and 7-day cultures of alveolar epithelial type 2 cells and alveolar macrophages in a linear, time and dose-dependent manner, with the rate of uptake being 15.3, 13.4 and 17.9 pmol/micrograms protein per 30 min, respectively. Uptake was not affected by metabolic inhibitors. The differential toxicity of the parent drug pentamidine, five analogues and six metabolites was assessed on freshly isolated and type 2 cells maintained in culture over 24 h. Toxicity, determined by the attachment ability of alkaline phosphatase positive cells containing lamellar bodies was greater in freshly isolated cells. Overall, three/four of the analogues proved less damaging to type 2 cells than the pentamidine with one derivative [1,3-bis(4-amidino-2-methoxy)propane], a compound particularly efficacious against pneumocystis in rats, showing minimal toxicity. Five metabolites (chain hydroxylated derivatives) were less toxic than the parent drug. However, one metabolite (N,N-dihydroxy derivative) was much more toxic than pentamidine to both type 2 cells and alveolar macrophages. It is concluded that as the type 2 cell can accumulate the drug, it represents a target cell which is particularly sensitive to pentamidine and/or some of its metabolites.

Diamine uptake by rat lung type II cells in vitro

Lung epithelial type II cells are responsible for synthesising and secreting pulmonary surfactant which reduces surface tension and prevents lung collapse. Type II cells replace type I cells and can proliferate in response to alveolar injury. An important aspect of this proliferation may be the ability of type II cells to accumulate amines actively, particularly the endogenous diamine putrescine. Putrescine is accumulated into isolated alveolar type II cells by an energy-dependent process. The uptake obeys saturation kinetics for which an apparent Km of 14.7 microM and Vmax of 130 pmol/micrograms DNA/hr was derived. The inhibitory effects of structurally similar amines on putrescine accumulation are described. As the herbicide paraquat has been suggested to share the same uptake system as putrescine from lung slice studies, this phenomenon was investigated in type II cell cultures. The results demonstrated that paraquat is a partially competitive inhibitor of putrescine accumulation in the cells. The Ki for the inhibition of putrescine uptake by paraquat in type II cells was calculated to be 69 microM, a value which closely matches the Km for paraquat (70 microM) predicted from lung slice studies. In molecular terms, the partial nature of the competition indicates that paraquat and putrescine do not occupy identical sites. Saturation of its site by paraquat reduced the affinity of putrescine 3.6-fold, but did not abolish it.

Extracellular biotransformation potential in mouse airways

The aims of this study were to determine if freshly isolated bronchiolar Clara cells retained biotransformation potential and whether components of phase 1 and/or phase 2 metabolism were present in the extracellular lining fluid of the lung airways. Approximately 550 microliters of lavage fluid was obtained from the mouse lung, which had been totally perfused of blood in order to facilitate the isolation of Clara cells from the same animal. Samples of acellular lavage fluid and aliquots of purified Clara cells were frozen at -20 degrees C prior to analysis. minimum numbers of cells and lavage fluid volumes, pooled from 2 CD-1 mice, were assayed for ethoxycoumarin-O-deethylase, NADPH-dependent cytochrome c reductase, glutathione-S-transferase(s) and non-protein sulphydryls (mostly reduced glutathione). Isolated Clara cells retained a high activity/level of all these parameters confirming their functional status for subsequent studies of xenobiotic metabolism in vitro. Acellular lavage fluid, from all the healthy animals, also contained mono-oxygenase, reductase and transferase activities and high non-protein sulphydryl levels suggesting that phase 1 and 2 reactions with xenobiotics could take place in the extracellular environment of the lung. Clara cells are known to undergo apocrine secretion and this removal of the apical cap containing secretory granules and smooth endoplasmic reticulum could account for the airway biotransformation potential that was observed.

Antioxidant Defences in the Extracellular Compartment of the Human Lung

Publisher Summary The external surface of the lung is covered with a thin layer of fluid, the respiratory tract lining fluid (RTLF). RTLF contains a wide range of compounds with distinct antioxidant properties. These antioxidants, especially the water-soluble compounds—glutathione, ascorbic acid, and uric acid—act as a first line of defense against inspired oxidants such as nitrogen dioxide and ozone. By reacting as sacrificial substrates with oxidizing gases, RTLF antioxidants protect the delicate outer surface of the lung. Studies in human volunteers have indicated that the distribution of antioxidants along the respiratory tract is not uniform. Uric acid is by far the most prevalent antioxidant in the nasal cavity while reduced glutathione is present in high concentrations in the lower airways. Marked differences in the amount of RTLF antioxidants among individuals have led to the proposal that those individuals with low RTLF antioxidant levels may be more susceptible to the impact of oxidizing pollutants.

Dose-dependent distribution of 3H-pentamidine following intra-tracheal administration to rats

1. The acute toxicity to the lung, and disposition, of pentamidine isethionate as a function of a pulmonary-delivered dose was investigated in the rat. 2. Acute toxicity 24 h following intra-tracheal instillation of pentamidine was determined by analysis of acellular surface protein concentration and differential cell counting of bronchoalveolar lavage fluid. These two parameters indicated that pentamidine doses > 10 mg/kg lead to increasingly severe oedematous and inflammatory responses within the lung. 3. Following intra-tracheal administration of sub-toxic doses of 3H-pentamidine (0.2-10 mg/kg), the extent of activity in liver, kidney, gut and lavage fluid at 24 h correlated significantly with dose, whereas the level of activity in lung was saturated at doses > 0.8 mg/kg. 4. Values of << 1 for liver:lung and kidney:lung ratios of 3H-activity at low pentamidine doses demonstrated the high affinity of the lung for the compound. These ratios substantially increased with pentamidine dose, reflecting distribution of the drug to liver and kidney. Association of radioactivity with these organs was rapid (< 30 min), and indicated that pentamidine is effectively absorbed from the respiratory tract following intra-tracheal instillation.