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.     

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.     

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.     

Changes in the pulmonary uptake and binding of drugs in an experimental model of lung fibrosis.

To investigate the effect of lung fibrosis on the pulmonary uptake and binding of drugs, rats were given weekly ip injections of 20 mg/kg paraquat dichloride for 6 weeks, and drug uptake and efflux in tissue slices from paraquatdamaged and control lungs were compared. Histologically, lungs from paraquat-treated animals showed multifocal areas of obliterative fibrosis, which appeared to involve 10–20% of the lung. Compared to controls, wet and dry weights of fibrotic lungs were increased 2.4- and 2.3-fold, respectively. Total lung protein per kilogram body weight was increased 85% in the paraquat-treated animals. Tissue/medium concentration ratios for [¹⁴C]benzylpenicillin in paraquat-damaged tissues were not significantly different from control values over a 15 to 240-min incubation period; the antibiotic did not appear to accumulate in lung slices. In contrast, [³H]isoniazid and [³H]dexamethasone were accumulated after 15 min, and uptake by paraquat-damaged tissues was significantly decreased (14–22%) for both drugs. In efflux studies with [³H]isoniazid and [³H]dexamethasone, slow-component rates for the drugs were not significantly different in control and fibrotic tissues; however, the fraction of the drugs that washed out at the slow component rate in the paraquat group was decreased 10–15% compared to controls. Differences in drug accumulation could not be accounted for by either a direct effect of residual lung paraquat on binding or metabolism by the lung. The results suggest that sites for drug accumulation in normal lung may be replaced by nonfunctional tissue in this model of lung fibrosis.     

1-Methyl-4-phenylpyridinium (MPP+) does not exhibit paraquat-like immunoreactivity

The structural analogy of paraquat with 1-methyl-4-phenylpyridinium (MPP+) has been implied in the aetiology of Parkinson's disease. The cross-reactivity of MPP+ to a specific antibody to paraquat was assessed by radioimmunoassay and was found to be very low. The results suggest that this polyclonal paraquat antibody does not mimic the MPP+ receptor.     

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.     

谷胱甘肽-S转移酶-中期因子融合蛋白原核表达及多克隆抗体的制备

目的 构建谷胱甘肽-S-转移酶(GST)和中期因子(MK)融合蛋白的原核表达质粒,并表达和纯化蛋白,制备多克隆抗体.方法 通过RT-PCR技术从人胃癌组织中扩增入MK编码序列,克隆入表达载体pGEX-1λT中,获得表达质粒pGEX-MK,并在大肠杆菌BL21 (DE3)中经IPTG诱导表达,通过亲和层析纯化表达的GST-MK融合蛋白,并以重组蛋白免疫兔子.结果 成功构建了GST-MK融合蛋白的原核表达载体,经诱导表达纯化得到GST-MK融合蛋白.免疫兔子后取多抗血清以间接ELISA检测效价达1∶64 000,Western blotting分析显示多克隆抗血清对MK蛋白特异结合.结论 MK在大肠杆菌中成功表达及其多克隆抗体的获得,为研究MK生物功能奠定了基础.     

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.     

Comparison of the tissue affinity of glucocorticoids to human lung, nasal and skin tissue in vitro.

High affinity binding of topically applied glucocorticoids to their target tissues is the basis for prolonged action of the drug and reduces efflux into the systemic circulation that might account for adverse effects. Since little information on the accumulation and depletion of glucocorticoids to tissues of therapeutic interest is available the binding behavior of different glucocorticoids to human lung, nasal and skin tissue is now evaluated and drug concentrations in different tissues are compared. Furthermore, the role of tissue lipids and proteins in glucocorticoid binding is investigated. Therefore, sliced human lung, nasal and skin tissues are incubated with glucocorticoid containing buffers and time course of adsorption and desorption is monitored. Two model glucocorticoids, the highly lipophilic fluticasone propionate (CAS 80474-14-2) and the rather hydrophilic hydrocortisone (CAS 50-23-7) are compared respecting their binding to native and lipid-depleted tissues. While total adsorption rates to different tissues were highly comparable for each glucocorticoid the observed initial desorption was clearly different. Highest initial depletion was seen for lung tissue, lowest for skin tissue. After initial depletion a prolonged desorption of very low concentrations is observed for all tissues. Lipid depletion of tissues did neither change accumulation not depletion behavior except that about twice as high concentrations were bound and depleted, probably due to protein denaturation. It is concluded that glucocorticoids primarily bind to protein components of human lung, nasal and skin tissues. Connective tissue proteins are discussed to bind glucocorticoids with higher affinity than other protein components, thus preventing high initial release rates. While total amounts of adsorption to different tissues are equivalent and initial desorption of glucocorticoids from saturated tissues varies, highest total remaining concentrations should be observed in skin tissue followed by nasal and lung tissue. Although tissue lipids seem to play no role in total glucocorticoid binding they are suggested to influence the rate constant of uptake and depletion.     

Isolation of substance P binding protein from rat brain.

Substance P (SP) binding protein of rat brain was solubilized by digitonin. The solubilized proteins were then purified by sequential gel filtration, concanavalin A lectin Sepharose, and SP-affinity chromatography. The calculated molecular weight of this purified SP binding protein was 76-74 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbits were immunized with the purified protein and resulting polyclonal anti-sera were tested. The immune serum significantly inhibited [3H]SP binding to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate solubilized membrane fractions from rat brain, whereas pre-bleed antiserum failed to inhibit the binding. This polyclonal antibody also inhibited the activity of 45Ca influx into astroglioma cells stimulated by SP, but does not inhibit that stimulated by histamine. Furthermore, this polyclonal antibody recognized the 76-74 kDa band as assessed by Western blotting. These data strongly suggest that this polyclonal antibody could recognize a part of the natural SP receptor site.     

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.     

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.     

Chronic toxicity, teratologic, and reproduction studies with hair dyes.

Administration of paraquat to mice, 1.67 and 3.35 mg/kg ip or 20 mg/kg po, daily on Days 8–16 of gestation induced no significant teratogenic effects, although a slight increase in nonossification of sternabrae was observed. Radioactivity reaching the mouse embryo after ip or po administration of [¹⁴C]paraquat on Day 11 of gestation was low. The fetal toxicity of diquat in rats, as measured by the number of dead and resorbing fetuses, was greater than that caused by paraquat after 15 mg/kg iv doses on various days of gestation, which correlated with higher fetal concentrations of [14C]diquat compared to [¹⁴C]paraquat. In perinatal organ distribution studies, more radioactivity from [¹⁴C]paraquat was retained in lung tissue of postnatal mice and rats than that in liver and kidney tissue. In prenatal studies, however, [¹⁴C]paraquat was retained in lung tissue of fetal rats after maternal administration of paraquat on Day 21 of gestation but not in lung tissue of fetal mice after maternal paraquat on Day 16 of gestation. This may be indicative of prenatal development of binding sites or of an active transport process for the uptake of paraquat into the lung or that elevated oxygen tensions in postnatal lungs contributes to paraquat retention in lung tissue.     

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 poisoning: an analytical toxicologic study of three cases.

Paraquat concentrations were measured in tissue, serum, urine and hemodialysate obtained from 3 patients who died 16.5 h, 22 days and 23 days after ingestion. In the patient who died 16.5 h post-ingestion, tissue paraquat levels were high. Kidney and liver had paraquat concentrations of 14 micrograms/g and 13.2 micrograms/g respectively, whereas lung tissue had a paraquat level of 3.8 micrograms/g. Low concentrations of paraquat were detectable in the tissues of the patients who died 22 and 23 days post-ingestion. Early in the poisoning, serum paraquat levels were high and large quantities of paraquat could be removed by both hemodialysis and forced diuresis. During an 8-h period, 713 mg of paraquat were removed by hemodialysis and 340 mg by forced diuresis. After the day of ingestion, little paraquat could be removed by hemodialysis or by forced diuresis; however, at all stages of the poisoning studied, hemodialysis was more effective than forced diuresis in removing paraquat from the blood.     

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.     

Inhibition of paraquat accumulation in rat lung slices by a component of rat plasma and a variety of drugs and endogenous amines

The accumulation of paraquat by slices of rat lung has been shown to be inhibited in vitro by the addition of rat plasma. For a given concentration of plasma, inhibition was constant with time and the amount of inhibition increased with increasing concentration of plasma. This suggests that there are components of rat plasma which inhibit paraquat accumulation by rat lung slices in a concentration-dependent manner. An ultrafiltrate of plasma also inhibited paraquat uptake, indicating that the inhibitor is a small molecular weight compound. A number of endogenous amines including noradrenaline, 5-hydroxytryptamine and histamine have been shown to reduce the concentration of paraquat accumulated into lung slices, as have several other drugs including imipramine, propranalol, burimamide and betazole. The relevance of these findings to the prevention of paraquat accumulation by lung is discussed.     

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.     

Mechanism of protection of alveolar type II cells against paraquat-induced cytotoxicity by deferoxamine

Paraquat toxicity has been associated with the generation of free radicals in alveolar epithelial cells in which paraquat specifically accumulates via a polyamine uptake system. In the present study we investigated whether deferoxamine (DF), an iron chelator that has antioxidant capacity and that also has a polyamine-like structure, could protect alveolar type II cells (ATTC) against injury by paraquat. Radiolabeled [3H]adenine ATTC were incubated in a medium containing 75 microM paraquat in the absence or presence of DF (500 microM). After 3 hr of incubation paraquat-mediated cytotoxicity of ATTC, as measured by [3H]adenine release, was significantly (P less than 0.005) decreased by addition of DF (26.6 +/- 2.6% vs 7.4 +/- 1.7%). Accumulation of radiolabeled [14C]paraquat at a concentration of 75 microM was also decreased (70%) by 500 microM DF from 94.8 +/- 2.1 to 28.9 +/- 6.7 nmoles paraquat/2.5 x 10(5) ATTC. This effect of DF was dose dependent and comparable with the protective effect of equimolar concentrations of putrescine. However, per cent uptake of paraquat at a concentration of 500 microM was not significantly inhibited by DF (1 mM), whereas paraquat-induced injury was still markedly reduced (36.2 +/- 2.5% vs 2.6 +/- 4.2%). This indicated that the protective effect of DF could not be explained by its competition with paraquat on uptake alone. In the same series of experiments using another iron chelator, pyridoxal benzoyl hydrazone (PBH), which has antioxidant properties similar to DF but does not show its polyamine-like structure, ATTC lysis was also prevented although paraquat uptake was not reduced. These in vitro data indicate that the mechanism of protection by DF against paraquat toxicity in lung epithelial type II cells is two-fold: inhibition of paraquat uptake through its compliance with the structural requirements necessary for transport, and inhibition of paraquat-induced iron-catalysed free radical generation.     

Comparison of one-electron reduction activity against the bipyridylium herbicides, paraquat and diquat, in microsomal and mitochondrial fractions of liver, lung and kidney (in vitro).

The first one-electron reduction steps of paraquat and diquat were compared using microsomal and mitochondrial fractions of rat liver, lung and kidney. Both fractions reduced each herbicide effectively, with the order of the Vmax values in microsomes and mitochondria being liver greater than lung greater than kidney and kidney greater than liver greater than lung, respectively. Although similar Vmax values were obtained from the liver and lung with the two subcellular fractions, the affinity of mitochondrial enzymes was lower, suggesting that the reduction of both herbicides in a microsomal site would be dominant in these two organs. The Vmax values for radical formation of paraquat were higher than those of diquat in all the endogenous one-electron reducing systems. The apparent Km values for diquat, however, were lower than those for paraquat in both subcellular fractions from the three tissues, indicating the superiority of the reduction for diquat to that for paraquat at low concentrations. This difference in the Km values supported the finding that the reduction velocity for diquat was significantly higher than that for paraquat at 1 mM concentration. Thus, at low concentrations, diquat would be reduced more easily than paraquat. In addition, tissue enzymatic specificity for paraquat was not obtained. From these data, it seems reasonable to conclude that the tissue-selective accumulation of paraquat previously proposed determines its toxicity.