Biomedical subjects
L L Smith
Publications and source records attributed to L L Smith.
Specific polyclonal and monoclonal antibody prevents paraquat accumulation into rat lung slices.
Sheep polyclonal and mouse monoclonal antibodies have been produced that bind to the bipyridyl herbicide, paraquat. The binding capacities and affinities of the various antibody solutions (serum, ascites, purified tissue culture supernatant) to paraquat were determined using a radioimmunoassay. All antibody solutions bound paraquat with high affinity (Ka = 10(9)-10(10) l/mol). The sheep polyclonal antisera, the mouse ascites fluid, and the purified culture supernatant had mean binding capacities of 8, 1 and 22 micrograms paraquat/ml respectively. All the antibody preparations were able to prevent the in vitro accumulation of paraquat into rat lung tissue. The amount of antibody to achieve this was dependent upon the binding capacity of the antibody solution, i.e. when the binding capacity of the antibody was equal to the amount of paraquat present in the incubation medium a total blockade of uptake was achieved. When antibody was added to lung tissue that had been accumulating paraquat for 1 hr, the inhibition of uptake was immediate and was complete for at least 2 hr. Both the radioimmunoassay and lung slice experiments indicate that an equivalent of 1 mg of IgG is required to bind 2.5 micrograms of paraquat ion. Preincubation of lung tissue with antibody did not affect the subsequent accumulation of paraquat, nor did it result in a detectable degree of intracellular neutralisation of paraquat as measured by paraquat's ability to stimulate the pentose phosphate pathway. The rate of efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was not increased by the presence of antibody in the incubation medium. In conclusion, neutralising antibodies to paraquat have been produced. They bind to paraquat in solution with high affinity and render the paraquat unavailable for its in vitro accumulation into lung cells.
Sterol hydroperoxide metabolism by Salmonella typhimurium.
In order to rationalize multiphasic dose-response data evincing mutagenicity towards Salmonella typhimurium TA1537 for sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide their metabolism by the bacterial test strain was investigated. The 5 alpha-hydroperoxide was isomerized to the 7 alpha-hydroperoxide and reduced to 5 alpha-cholest-6-ene-3 beta,5-diol; the 7 alpha-hydroperoxide was reduced to cholest-5-ene-3 beta,7 alpha-diol and transformed to 3 beta-hydroxycholest-5-en-7-one. The 3 beta,5 alpha-diol and 3 beta,7 alpha-diol were not interconverted nor was either transformed to the 7-ketone.
Putrescine and 5-hydroxytryptamine accumulation in rat lung slices: cellular localization and responses to cell-specific lung injury.
The cellular localization of putrescine (1,4-diaminobutane) and 5-hydroxytryptamine (5HT) following the accumulation of tritium-labeled putrescine (2.5 microM) or 5HT (0.5 microM) into rat lung slices was determined by autoradiography at the light microscope level. Putrescine labeling was found to occur in type II alveolar epithelial cells and in branchiolar nonciliated (Clara) cells, and possibly also in type I alveolar epithelial cells. The pattern of 5HT labeling was clearly different from that with putrescine, since the parenchyma was diffusely labeled with no preferential location in type II cells, but with strong labeling of the endothelium of large vessels and also the pleural mesothelium. The apparent kinetic parameters for the tissue uptake of [3H]putrescine (2.5 to 80 microM) and [14C]5HT (0.5 to 16 microM; both being simultaneously present in a 5 to 1 molar ratio) were studied in lung slices from normal rats and rats pretreated with O,S,S-trimethyl phosphorodithioate (OSSMe, 11 to 95 mg/kg, po), with paraquat (20 mg/kg, ip), or with alpha-naphthylthiourea (ANTU, 5 or 10 mg/kg, ip). OSSMe and paraquat were used as models for pulmonary epithelium-damaging agents, and ANTU was taken as a model for a pulmonary endothelium-damaging agent. The Vmax for the uptake of 5HT was significantly increased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of 5HT was unchanged (5 mg/kg) or increased (10 mg/kg, Km also increased). These results indicate that in lung slices the response to lung injury may be associated with an increased accumulation of 5HT. The Vmax for the uptake of putrescine was significantly decreased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of putrescine was unchanged (5 mg/kg) or decreased (10 mg/kg, no change in Km). These results suggest that a decreased putrescine uptake is a sensitive index of pulmonary epithelial damage.
Procedures for enhancing the utility of the metallothionein promoter for the regulated expression of downstream open reading frames.
Procedures which enhance the inducibility of the mouse metallothionein I (mMT-I) transcriptional promoter in mouse C127 cells transformed by bovine papilloma virus have been investigated. These include: (i) induction with Zn2+ at low serum concentration, and (ii) use of a 'superinduction' protocol (presence of 1 microgram/ml of cycloheximide during induction with Zn2+, followed by 2 micrograms/ml of actinomycin D). Use of procedure (i) alone gave a 15- to 20-fold induction of expression of a downstream open reading frame (ORF), which is comparable to the maximum inducibility achieved with mMT-I in other systems. Use of procedures (i) and (ii) in combination allowed a 50-fold induction. Three different reporter ORFs (rabbit ferritin L subunit, human chorionic gonadotropin alpha subunit, and human lutropin beta subunit), in three different chromosomal contexts, responded to these procedures. The maximum rate of expression achieved was estimated at over 10(9) molecules per cell per day, which is 20% of the transformed cell's protein synthetic capacity. At these extremely high levels some of the induced products were cytotoxic.
Can carotid duplex scanning supplant arteriography in patients with focal carotid territory symptoms?
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Operative balloon angioplasty in the treatment of internal carotid artery fibromuscular dysplasia.
Seven patients with symptomatic fibromuscular dysplasia have had eight internal carotid arteries treated by operative balloon dilatation. This technique is described in detail and provides three distinct advantages over conventional graduated intraluminal dilatation--atraumatic passage of the catheter through the affected vessel with fluoroscopic guidance; precise dilatation of the involved segment of the internal carotid artery; and the application of a radial force against the arterial wall rather than a longitudinal shear force, thereby making intimal damage less likely. There were no treatment complications in this group of patients. This technique of balloon angioplasty is compared with the results of treatment in eight patients with fibromuscular dysplasia of the internal carotid artery, who had percutaneous transfemoral angioplasty. Long-term follow-up revealed the resolution of symptoms in all patients.
Mechanism of paraquat toxicity in lung and its relevance to treatment.
The symptoms of paraquat poisoning depend largely on the amount of compound consumed, although in many cases the most characteristic feature of poisoning is lung damage, causing severe anoxia which leads to death. Studies in experimental animals have demonstrated that paraquat produces an acute damaging phase in the lung, followed by a reparative phase dominated by an extensive fibrosis. The latter is a major contributor to the lung lesion that causes anoxia. The specific toxicity in the lung can be explained in part by the selective accumulation of paraquat into this organ in comparison with other tissues. The accumulation is energy-dependent and probably specific to certain lung cells. It is now known that paraquat is accumulated into the lung by a recently described diamine transport process located in the alveolar epithelial cells and the Clara cells of the airways. When accumulated, paraquat undergoes a NADPH-dependent one-electron reduction to for its free radical which almost instantly reacts with molecular oxygen to reform the cation and concomitantly produce superoxide anion. This species of oxygen radical can contribute to the formation of more toxic species of radical which may directly damage vital cellular constituents. Paraquat has been shown to stimulate rapidly the pentose phosphate pathway and inhibit the synthesis of fatty acids in the lung in a dose-dependent manner. In addition there is a rapid increase in the pulmonary levels of mixed disulphides and the eventual reduction of NADPH levels in the lung. These results are consistent with the suggestion that paraquat causes a rapid and pronounced oxidation of NADPH which initiates compensatory biochemical responses in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)
The development of mouse monoclonal antibodies that bind and neutralize paraquat.
Mouse hybridomas have been produced that produce antibody that can bind to the bipyridyl herbicide paraquat with high affinity (Ka = 1.2 X 10(10) litres/mole). In addition, one cell line, 321, secretes antibody in the concentration range 60-100 micrograms/ml of culture, and 5 mg/ml in ascites fluid. Such high concentrations make it feasible to produce gram quantities of antibody with relative ease and low cost. The antibody is of class and sub-class IgG1 and can be readily purified by protein A and paraquat specific affinity chromatography. Radioimmunoassays and in vitro neutralization studies have demonstrated that 1 mg of antibody is required to bind 2.5 micrograms of paraquat. When antibody binds to paraquat in vitro it renders the paraquat unavailable for accumulation into rat lung tissue.
Morphological and biochemical correlates of chemical induced injury in the lung. A discussion.
We have reviewed some of the factors which contribute to lung damage by various toxicants. These include disposition of the chemical, its metabolism, individual cell type susceptibility and the potential for the tissue to repair. We have discussed the use of biochemical parameters to measure the functional activity of individual cell types in order to predict the damage to specific cell types and concluded that careful morphological analysis of lung tissue is likely to provide a more sensitive and informative measure of specific cell type injury. However, in order to investigate the mechanism of toxicity of pulmonary toxicants it is essential to establish the primary biochemical event that leads to cell damage and morphological change. The importance of separating the relevant biochemical change(s) from the cascade of biochemical events associated with dead and dying cells and the reparative response of the lung is emphasised.
Mutagenic sterol hydroperoxides.
Sterol hydroperoxides 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide and 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide show weak dose-response direct mutagenicity towards Salmonella typhimurium strain TA 1537 in a liquid medium incubation bioassay. Responses were compromised by metabolism of the sterol hydroperoxides and by phase separation during the incubation period. Mutagenicity responses were increased by added superoxide dismutase but diminished by added rat liver S9 enzymes and abolished by added catalase. Catalase also abolished the stimulatory effect of superoxide dismutase. These results indicate that superoxide and peroxide be implicated in the mutagenicity responses.
The response of the lung to foreign compounds that produce free radicals.
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High-performance liquid chromatography of linoleic acid hydroperoxides and their corresponding alcohol derivatives.
The four major hydroperoxides derived from autoxidation or lipoxygenase action on linoleic acid or on methyl linoleate and their corresponding alcohol derivatives are resolved by high-performance liquid chromatography on Zorbax SIL 3 micron particulate columns irrigated with hexane-diethyl ether-acetic acid mixtures. The four major linoleic acid hydroperoxides are interconverted at different rates in benzene or carbon tetrachloride solutions but are stable to storage under nitrogen at -70 degrees C for several months.
Paraquat toxicity.
Paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride) is marketed as a contact herbicide. Although it has proved safe in use there have been a number of cases of poisoning after the intentional swallowing of the commercial product. The most characteristic feature of poisoning is lung damage, which causes severe anoxia and may lead to death. The specific toxicity to the lung can be explained in part by the accumulation of paraquat into the alveolar type I and type II epithelial cells by a process that has been shown to accumulate endogenous diamines and polyamines. When accumulated, paraquat undergoes an NADPH-dependent, one-electron reduction to form its free radical, which then reacts avidly with molecular oxygen to reform the cation and produce superoxide anion, which in turn will dismutate to form H2O2. This may lead to the formation of more reactive (and hence toxic) radicals which have the potential to cause lipid peroxidation and lead to cell death. Biochemical changes provoked by paraquat in the lung suggest that it causes a rapid, pronounced and prolonged oxidation of NADPH that initiates compensatory biochemical processes in the lung. NADPH may be further depleted as it is consumed in an attempt to detoxify H2O2 or lipid hydroperoxides. Thus it is possible that with toxic levels of paraquat in the cell, compensatory biochemical processes are insufficient to maintain levels of NADPH consistent either with cell survival or with the ability to detoxify H2O2 or prevent lipid peroxidation.
Pulmonary accumulation of methylglyoxal-bis(guanylhydrazone) by the oligoamine uptake system.
The accumulation of methylglyoxal-bis(guanylhydrazone) (MGBG) into rat lung slices and its relationship to the accumulation of oligoamines has been investigated. MGBG was accumulated by rat lung slices by a process which obeyed saturation kinetics (Km 6.6 microM; Vmax 75.3 nmoles/g wet wt lung/hr). The uptake process appeared to be identical to those described for the accumulation of oligoamines and paraquat, being both KCN-(1 mM) and temperature-sensitive but insensitive to ouabain (100 microM). Pulmonary MGBG accumulation was found to be sodium-independent, either being enhanced or unaffected by sodium chloride-deficient media, so distinguishing the process from that described for the monoamine, 5-hydroxytryptamine. The ability and nature of various rat tissue slices to accumulate MGBG generally followed that of the oligoamines. Slices of lung, brain cortex and seminal vesicles accumulated MGBG by a KCN-sensitive and temperature-dependent process. These observations, together with the ability of MGBG to inhibit pulmonary oligoamine accumulation, indicate that it is the uptake system for the oligoamines which is mainly responsible for the in vitro accumulation of MGBG.
High-performance liquid chromatography of cardiolipin.
Resolution of freshly prepared and of commercially available (degraded) samples of cardiolipin into 15-30 components has been accomplished by reversed-phase high-performance liquid chromatography using a 3-micron particulate Microsorb C18 column irrigated with linear gradients of acetonitrile--methanol--10 mM phosphate buffer pH 7.4. Selected resolved components were crystallized and characterized by infrared absorption spectra. Saponification of other components and identification of component fatty acids by reversed-phase high-performance liquid chromatography demonstrated the presence of ten fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4), with linoleic acid (18:2) identified in all resolved components. From fatty acid composition data it appears that several resolved fractions consist of single cardiolipin molecular species.
Improved high-performance liquid chromatography of sphingomyelin.
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Differences in naphthalene-induced toxicity in the mouse and rat.
Following the intraperitoneal administration of naphthalene (200 mg/kg) to mice, the lung, in comparison with other organs, was selectively damaged. Histological examination of the lungs showed that it was the non-ciliated, bronchiolar epithelial cells (Clara cells) which were damaged. At higher doses (400 mg/kg and 600 mg/kg, i.p.), there was also damage to the cells in the proximal tubules of the kidney. In contrast to the effect in mice, doses of naphthalene as high as 1600 mg/kg (i.p.) caused no detectable pulmonary or renal damage in the rat. This difference in toxicity between the mouse and rat was reflected by the ability of naphthalene to more severely deplete the non-protein sulphydryls in the mouse lung and kidney than in those organs in the rat. In order to investigate the species difference in toxicity, the metabolism of naphthalene by lung and liver microsomes of the mouse and rat was studied. In all cases, naphthalene was metabolised to a covalently bound product(s) and to two major methanol-soluble products, which co-chromatographed with 1-naphthol and 1,2-dihydro-1,2-dihydroxynaphthalene. However, both the covalent binding and metabolism were approximately 10-fold greater in microsomes prepared from mouse lung compared with those from the rat. This observation may in part explain the difference in toxicity of naphthalene to the mouse and rat lung. As 1-naphthol is a major metabolite of naphthalene and previous work had suggested that most of the microsomal catalysed binding of naphthalene was due to further oxidation of 1-naphthol, the role of 1-naphthol in mediating the naphthalene-induced toxicity was investigated. In neither the mouse nor the rat did 1-naphthol cause a depletion of non-protein sulphydryl levels or tissue damage in the liver, lung or kidney. Thus the toxicity of naphthalene does not appear to be mediated via 1-naphthol.