The relationship between 5-hydroxytryptamine and paraquat accumulation into rat lung

The uptake of 5-hydroxytryptamine (5HT) into rat lung slices has been shown to obey saturation kinetics and to be inhibited by imipramine, metabolic inhibitors and a sodium-free medium. The apparent K_m for the uptake process was found to be 3.3 μMwith a V_max of 6 nmoles/g wet wt/min. Lung slices taken from rats given a dose of paraquat known to damage type I and type II lung epithelial cells showed inhibition of paraquat uptake but no inhibition of 5HT uptake. This together with the stimulation of paraquat accumulation into rat lung slices in a sodium-free medium leads to the conclusion that the uptake of paraquat and 5HT into the lung does not occur in the same cell type.     

Paraquat accumulation: Tissue and species specificity

Rat tissues have been examined in vitro for their ability to accumulate paraquat or diquat to concentrations in excess of those present in the incubation medium. With a concentration of 10^?6 M. lung slices were able to accumulate paraquat to concentrations nearly ten times that of the medium. and brain slices to concentrations double that of the medium, over a period of two hours. Neither slices of lung nor brain accumulated diquat significantly from a concentration in the medium of 10^?6 M. The accumulation of paraquat by brain slices, like that of lung slices, has been shown to he energy-dependent. Other organs examined showed little, if any, ability to accumulate either paraquat or diquat. Lung slices from dog. monkey and rabbit have also been shown to possess the ability to accumulate paraquat in vitro. After oral dosing of paraquat to rats. the lung concentration increased with time to six times that of the plasma after 30 hr. Other organs, with the exception of the kidney, did not concentrate paraquat to the same extent. Kidney concentrations after oral dosing of both paraquat and diquat were high throughout the period of time studied. It is. therefore, suggested that the apparent selectivity exhibited by paraquat for the lung is associated with the accumulation process.     

Uptake and efflux of 14C-paraquat by rat lung slices: the effect of imipramine and other drugs.

Paraquat accumulation by rat lung slices incubated at 10 or 100 μm concentration was linear with time and the accumulated paraquat was “noneffluxable.” Imipramine (100 or 500 μm) inhibited paraquat (10 μm) uptake by 38 and 85%, respectively, and 500 μm imipramine enhanced paraquat efflux by 40%. The combination of impaired uptake and enhanced efflux suggested the possibility that imipramine might reduce the toxicity of paraquat in intact animals. However, at the doses used, imipramine did not alter paraquat toxicity in vivo. Eleven other drugs were shown to inhibit uptake, but only five enhanced paraquat efflux.     

The acute toxic effects of paraquat and diquat on the rat kidney.

The bipyridyls, paraquat and diquat, cause mild renal tubular damage in the rat. A marked diuresis, albuminuria, glucosuria, and an increased plasma urea concentration occurred 6–24 hr after paraquat (680, po, or 108 μmol/kg, sc) and an increased urinary excretion of N-acetyl-β-d-glucosaminidase occurred 6–24 hr after paraquat (680 μmol/kg, po). Diquat (680 μmol/kg, po) produced proteinuria and glucosuria 6–24 hr after dosing. Histological examination of the kidneys showed there was mild hydropic degeneration of the proximal convoluted tubules. Mercuric chloride (HgCl₂), a nephrotoxic agent, was used as a positive control in these studies dosed at 7.4 μmol/kg, sc, and produced marked tubular necrosis. Biochemical studies showed that both paraquat and diquat decrease N′-methylnicotinamide (NMN) but not p-aminohippurate (PAH) accumulation by renal cortical slices when added in vitro, suggesting competition for the base transport system. However, no decrease in NMN or PAH accumulation was seen in renal cortical slices from rats treated with paraquat or diquat. Both bipyridyls stimulated pentose phosphate pathway and inhibited fatty acid synthesis when added in vitro to renal cortical slices. However, neither of these parameters were affected in renal cortical slices prepared from rats treated with paraquat or diquat. We conclude that both bipyridyls cause renal tubular damage but this damage is mild compared with that seen after HgCl₂. And that biochemical indicators of paraquat damage in the lung are not altered in renal cortical slices from bipyridyl-treated rats. These minimal changes observed using histological and biochemical techniques contrast sharply with the effects these compounds have on renal excretory function.     

The relevance of pentose phosphate pathway stimulation in rat lung to the mechanism of paraquat toxicity

Paraquat and diquat have been shown to stimulate the production of ¹⁴CO₂ from [1-¹⁴C]-glucose by slices of rat lung, but not the production of ¹⁴CO₂ from [6-¹⁴C]glucose. This indicates stimulation of the pentose phosphate pathway. Paraquat was effective at concentrations as low as 7.5 × 10^?7 M whilst a concentration of diquat of 10^?5 M was required for comparable stimulation. Maximal stimulation occurred with approximately 10^?5 M paraquat and approximately 10^?4 M diquat. The stimulation of pentose phosphate pathway in lung slices by paraquat has been shown to be related to paraquat accumulation.Lung slices from rats dosed intravenously with 65 μmoles of either paraquat or diquat/kg body wt had increased pentose phosphate pathway activity compared with slices from saline injected controls. At all times studied from 0.5 to 18 hr after injection, pentose phosphate pathway activity in slices from diquat poisoned rats was equal to or greater than that observed in slices from paraquat poisoned rats. Since only rats dosed intravenously with paraquat subsequently develop lung damage, it is concluded that there is no simple relationship between stimulation of the pentose phosphate pathway in lung and the production of lung damage.     

Factors affecting the efflux of paraquat from rat lung slices

In an attempt to reduce the toxicity of paraquat several compounds were examined for their ability to increase the rate of efflux of paraquat from the lung. The compounds were selected because they were known, from in vitro studies, to reduce the accumulation of paraquat into the lung. Histamine (100 μM), promethazine (100 μM), putrescine (100 μM), bromthymol blue (300 μM) and the metabolic inhibitors iodoacetate (1 mM), rotenone (100 μM) and KCN (1 mM) have been shown to reduce the accumulation of paraquat into rat lung slices, as did the incubation of slices under nitrogen.The efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was biphasic, having a very fast component and a slow component. The slow component was first order and was characterised by a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 17 h. This half life is similar to that seen in vivo (24h) following intravenous dosing. When lung slices prepared from rats dosed intravenously with paraquat were incubated in the presence of iodoacetate (1 mM) or under nitrogen, the half life of paraquat in the slices was reduced to approximately 3 h. In the presence of rotenone (100 μM) it was reduced to approximately9 h. Histamine (100 μM) and promethazine (100 μM) did not affect the efflux of paraquat from lung slices. Bromthymol blue, a dye which forms “ion pair” complexes with paraquat, also significantly increased the efflux of paraquat from lung slices. The effect of bromthymol blue, however, decreased with time and thus paraquat efflux in the presence of bromthymol blue did not obey first order kinetics.In order to measure cellular viability of lung slices, oxygen comsumption, glucose oxidation and the rate of the efflux of protein from the slices into the incubation medium were determined. Iodoacetate (1 mM) and rotenone (100 μM) almost abolished oxygen consumption and glucose oxidation whereas these activities were inhibited to a lesser extent by bromthymol blue (300 μM) (18% and 30%, respectively). During the first 30 min of incubation in the presence of KCN (1 mM) oxygen consumption was almost abolished but between 30 min and 4 h returned to control levels. The effect of KCN could therefore be divided into 2 phases. Over 4 h incubation glucose oxidation was inhibited by 36%. Iodoacetate (1 mM) and incubation under nitrogen caused the most pronounced increases in the rate of protein efflux from slices. KCN (1 mM) and rotenone (100 μM) also increased the rate of protein efflux but to a lesser extent. We have therefore suggested that the effect of KCN (1 mM) on cellular viability, while severe, may be less than that of iodoacetate (1 mM) or incubation under nitrogen.We have concluded from these studies that: (1) the reduction in the accumulation of paraquat or increase in its efflux produced by metabolic inhibitors may be a consequence of lung cell damage; (2) bromthymol blue and putrescine cause an increase in the efflux of paraquat from the lung in vitro without damaging the tissue suggesting that in principle this approach may be useful in vivo; and (3) histamine and promethazine reduce the uptake of paraquat into the lung by direct competition with paraquat since they neither increase the rate of efflux of paraquat from the lung nor reduce cellular viability.     

The relationship between paraquat accumulation and covalent binding in rat lung slices

The accumulation and covalent binding of paraquat in rat lung slices were both linear for 6 hr in room air incubations. Binding continued to increase in slices transferred to paraquat-free buffer after 3 hr of incubation in paraquat although accumulated paraquat decreased. Binding in 100% O2 was decreased slightly. Active accumulation in 100% N2 did not occur, but binding proceeded at one-third the rate observed in room air. Ascorbate decreased accumulation in room air, although binding was unaffected. Reductants had no effect on binding in 100% nitrogen. Paraquat binding in slices of various organs was in the order of lung greater than liver greater than heart greater than kidney cortex. Mitochondrial proteins were found to have the highest concentration of bound paraquat in lung slices followed in order by microsomal protein greater than nuclear protein = cytosolic protein. The binding of paraquat is postulated to involve a reduced species, presumably the monovalent radical.     

Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices

 The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been studied in precision-cut rat lung slice cultures. Lung slices were prepared from male Sprague-Dawley rats using an agarose gel instilling technique with a Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene or paraquat produced concentration dependent decreases in lung slice protein synthesis and potassium content. EC₅₀ values (concentration to produce a 50% inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat, 1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate that precision-cut lung slices are a useful in vitro model system for studying the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a rapid in vitro screen for identifying pulmonary toxicants and to evaluate species differences in response. Received: 10 October 1994 / Accepted: 30 November 1994     

The accumulation of diamines and polyamines into rat lung slices

The diamine cadaverine, and the polyamines spermidine and spermine have been shown to accumulate into rat lung slices by an uptake process which obeyed saturation kinetics. The apparent K_m values for the accumulation process of cadaverine, spermidine and spermine were 19, 11 and 15 μM respectively with V_max values of 937, 768 and 617 nmoles/g wet weight/hr respectively. The accumulation was KCN sensitive, indicative of an energy dependent process, although spermine did show some non-specific binding to lung tissue. Cadaverine, spermidine and spermine were not accumulated by slices of liver, kidney, heart and spleen to concentrations much greater than that in the medium. They were accumulated, however, by a KCN sensitive process into brain slices although the accumulation was much less than that which occurred in lung slices. The diamine, putrescine, exhibited a concentration-dependent inhibition of the ability of lung slices to accumulate cadaverine and the polyamines. These data have led us to conclude that the transport process in the lung, which has recently been shown to accumulate the diamine putrescine, is also capable of accumulating cadaverine, spermidine and spermine. Thus, by analogy with putrescine, there exists in specific lung cells a membrane receptor(s) which is selective in its acceptance and transport of diamines and polyamines.     

Structure-activity correlations of amines inhibiting active uptake of paraquat (Methyl viologen) into rat lung slices

Analysis of amine structure with respect to inhibitory potency utilized a new method for determining equipotent inhibitor concentrations of paraquat uptake by lung slices. Fifteen N-alkyl homologues of paraquat (viologens) were tested and inhibition of lung uptake of paraquat was found to be a function of the inductive effect and steric bulk of groups attached to the nitrogens of 4,4′-bipyridyl. Several classes of amine inhibitors were examined. Polyamines were generally more potent than compounds containing only one quaternizable nitrogen at pH 7.4. α, ω-Diaminoalkanes were the most potent inhibitors of paraquat accumulation by lung slices.     

Binding of [methyl-3H]paraquat to rat,rabbit, hamster,mouse and guinea pig lung proteins,in vitro

When incubated with the rat lung slices [methyl-³H] paraquat was found to bind covalently to acid-insoluble proteins. The evidence in the present study indicated that the binding of radioactivity was not due to the formation of a reactive metabolite secondary to mixed-function oxidase (MFO)-mediated bioactivation of paraquat but was probably due to formation of paraquat free radicals. Preincubation of lung slices with CN?, 2,4-dinitrophenol (DNP), p-hydroxymercuribenzoate and cysteine inhibited binding, while anaerobic conditions has a marked stimulatory effect. There were no significant differences in the extent of covalent binding of [methyl-³H]paraquat to lung protein of rat, rabbit, mouse, guinea pig, and hamster.     

Ascorbic acid and paraquat: Oxygen depletion with concurrent oxygen activation

Ascorbic acid and paraquat produce an efficient redox pair which will deplete oxygen from physiological buffer systems. This reaction is partially blocked by superoxide dismutase or catalase and is potentiated by the hydroxyl radical scavenger, dimethyl sulfoxide. Mitochondria isolated after incubation of rat lung slices with 1.0 mm paraquat and 10.0 mm ascorbate were unresponsive to ADP (were “uncoupled”). Also, metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose was inhibited by 50% in lung slice preparations. These results suggest a synergistic interaction of ascorbate and paraquat which results in disruption of subcellular energy metabolism. Paraquat accumulation into lung slices, an active transport process of the pulmonary cell membrane, was also inhibited by the addition of ascorbate. These results suggest that the previously reported in vivo potentiation of paraquat toxicity by ascorbate may be related to either: (1) a decreased subcellular oxygen availability, or (2) the presence of activated oxygen species, or (3) both.     

The effect of chlorpromazine on the uptake and efflux of paraquat in rat lung slices

The uptake of paraquat by rat lung slices was inhibited by chlorpromazine in a concentration- and time-dependent manner. In addition, the efflux of paraquat from lung slices was enhanced by chlorpromazine in a similar fashion. These in vitro findings suggested that chlorpromazine might be useful in vivo in reducing pulmonary paraquat content and, in turn, its pneumotoxicity. However, chlorpromazine potentiated the lethal toxicity of paraquat rather than ameliorating it. This potentiation by chlorpromazine was found to correlate with a reduction in the urinary excretion of paraquat and concomitant increase in pulmonary paraquat concentrations.     

In vitro analysis of the renal handling of paraquat.

Accumulation of paraquat by mouse renal cortical slices was related to the concentration of paraquat in the medium and the duration of incubation. Paraquat accumulation was depressed by incubation of slices under nitrogen or by addition of metabolic inhibitors. Accumulation of a second organic base, N-methylnicotinamide (NMN), was depressed by a concentration of paraquat which failed to influence accumulation of the organic acid, p-aminohippurate (PAH). The uptake component of NMN accumulation was inhibited by paraquat. The data suggest that paraquat is accumulated by an energy-requiring process and that this accumulation occurs via the organic base transport system. In addition, an apparently toxic effect of paraquat on cortical slice function was observed when the incubation temperature was raised from 25 to 37°C. At this temperature, 10^?3m paraquat depressed not only NMN accumulation but PAH accumulation and slice oxygen consumption as well. Thus, paraquat can be toxic to the function of kidney slices and this effect appears to be temperature-dependent.     

Potentiation of the cell specific toxicity of paraquat by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Implications for the heterogeneous distribution of glutathione (GSH) in rat lung

In order to study oxidative stress in the lung, we have developed a rat lung slice model with compromised oxidative defences. Lung slices with markedly inhibited glutathione reductase activity (approximately 80% inhibition) were prepared by incubating slices, with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 microM) in an amino acid-rich medium for 45 min at 37 degrees. These lung slices had similar levels of GSH and ATP and polyamine uptake (a marker of alveolar epithelial type I and II cell function) to control rat lung slices. We have utilized these BCNU pretreated slices to study the effects of the herbicide, paraquat, in comparison to those of 2,3-dimethoxy-1,4-naphthoquinone, a potent redox cycler. Paraquat (10-100 microM) caused only minimal changes in the levels of GSH or ATP in control or compromised slices. In contrast, 2,3-dimethoxy-1,4-naphthoquinone caused a decrease in GSH in control slices but a markedly enhanced decrease in both GSH and ATP in compromised slices. Both compounds had only limited effects on putrescine and spermidine uptake in control slices. However, they caused a marked inhibition in compromised slices. Paraquat had little effect on 5-hydroxytryptamine uptake (a marker of endothelial cell function) in either control or compromised slices whereas the quinone inhibited uptake in the compromised slices. Thus, the lack of effect of paraquat on GSH and ATP does not support the involvement of oxidative stress in its toxicity. In contrast, using polyamine uptake, as a functional marker of alveolar epithelial cell damage, suggests a role for redox cycling. As paraquat is known to be accumulated primarily in alveolar type I and II cells (a small fraction of the lung cell population), our data suggest that only a small proportion of pulmonary GSH and ATP is present in alveolar epithelial type I and II cells but that much larger amounts may be present in endothelial cells. These studies highlight the problem of gross tissue measurements in heterogeneous tissues such as the lung.     

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.     

Glucose ameliorates the depletion of NADPH by paraquat in rat lung slices

Paraquat-stimulated NADPH depletion in rat lung slices is responsive to exogenous glucose concentration. Lung slices incubated with 11 mM glucose and 10(-4) M paraquat had a 40% lower NADPH/NADP+ ratio than did control lung slices. Incubation with no added glucose and 10(-5) M paraquat caused a 41% decrease in NADPH/NADP+. With paraquat at 10(-5) M, glucose at 1.1, 5.5, 11, or 22 mM increased NADPH/NADP+ ratios in a concentration-dependent manner until, at 22 mM glucose, the effect of paraquat was prevented. The sum of NADP+ plus NADPH was only 60% of control with 10(-5) M paraquat and no glucose. However, with any concentration of glucose from 1.1 to 22 mM, the total was 92% of control. The results indicate that glucose may be beneficial in preventing paraquat-mediated NADPH depletion in rat lung slices.     

The accumulation of cystamine and its metabolism to taurine in rat lung slices

The objective of these studies was to determine the accumulation and fate of the disulphide, cystamine by rat lung slices. Cystamine was accumulated by two active uptake systems that obeyed saturation kinetics, with apparent Km values of 12 and 503 microM, and maximal rates of 530 and 5900 nmol/g wet weight/hr respectively. The high affinity system was competitively inhibited by the diamine, putrescine and the herbicide paraquat, which are themselves accumulated. Thus, this pulmonary uptake process appears to be identical for all three compounds. In contrast, the low affinity process was not inhibited by putrescine, and this process results from the diffusion of cystamine into the cell and its subsequent metabolism. Upon accumulation, cystamine was metabolised, predominantly to the sulphonic acid, taurine, with 10-20% of the intracellular label covalently binding to protein. Conversion to taurine was unaffected by amine oxidase inhibitors, but was decreased after GSH depletion, suggesting that pulmonary cystamine metabolism is glutathione-dependent, and is not mediated by diamine oxidase. Both cystamine and taurine have been implicated as antioxidants, and we suggest that cystamine is actively accumulated by the lung as part of the process to protect pulmonary tissue against oxidative stress.     

Kinetics of paraquat in the isolated rat lung: Influence of sodium depletion

Paraquat accumulates in the lung through a characteristic polyamine uptake system. It has been previously shown that paraquat uptake can be significantly prevented if extracellular sodium (Na+) is reduced, although the available data correspond to experiments performed using tissue slices or incubated cells. This type of in vitro study fails to give information on the actual behaviour occurring in vivo since the anatomy and physiology of the studied tissue is disrupted. Accordingly, the aim of the present study was to explore the usefulness of the isolated rat lung model when applied to characterize the kinetic behaviour of paraquat in this tissue after bolus injection under standard experimental conditions as well as to evaluate the influence of iso-osmotic replacement of Na+ by lithium (Li+) in the perfusion medium. The obtained results show that the present isolated rat lung model is useful for the analysis of paraquat toxicokinetics, which is reported herein for the first time. It was also observed that Na+ depletion in the perfusion medium leads to a decreased uptake of paraquat in the isolated rat lung, although it seems that this condition does not contribute to improve the elimination of paraquat once the herbicide reaches the extravascular structures of the tissue, since the paraquat tissue wash-out phase is similar under both experimental conditions assayed.     

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.