Ian Wyatt

Paraquat accumulation: Tissue and species specificity

Abstract 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.

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

Abstract Paraquat and diquat have been shown to stimulate the production of 14 CO 2 from [1- 14 C]-glucose by slices of rat lung, but not the production of 14 CO 2 from [6- 14 C]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.

The accumulation of putrescine into slices of rat lung and brain and its relationship to the accumulation of paraquat

Abstract The diamine putrescine inhibits accumulation of the herbicide paraquat into slices of rat lung. Putrescine was also shown to be accumulated into rat lung slices by an uptake process which obeyed saturation kinetics. The apparent K m for the process was 7 μM with a V max of 330 nmoles/g wet wt of lung/hr. The uptake of putrescine was enhanced when the slices were incubated in sodium deficient medium and was inhibited by iodoacetate (1 mM) together with KCN (1 mM), rotenone (100 μM), and by paraquat. Putrescine was not accumulated by slices of liver, kidney, heart or spleen to concentrations much above that in the medium. It was accumulated however by a KCN sensitive process into brain slices although the accumulation was less than that which occurred in lung slices. Lung slices taken from rats given an amount of paraquat known to damage both type I and type II lung alveolar epithelial cells were less able to accumulate putrescine or paraquat than lung slices taken from control rats. This reduction in uptake was similar for both compounds. These data have led us to conclude that (1) the process in the lung and brain which accumulates paraquat is that which is normally responsible for the uptake of putrescine in particular and diamines in general, and (2) it is the alveolar type I and type II cells of the lung which possess a receptor for the active uptake of the diamine putrescine.

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 Km values for the accumulation process of cadaverine, spermidine and spermine were 19, 11 and 15 microM respectively with Vmax 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.

The accumulation and localisation of putrescine, spermidine, spermine and paraquat in the rat lung: In vitro and in vivo studies

Putrescine was accumulated into the isolated perfused rat lung by a temperature dependent process. The uptake obeyed saturation kinetics for which an apparent Km of 14 microM and Vmax of 48 nmol/g wet wt/hr was derived. After rats were dosed subcutaneously with [14C]putrescine, it was accumulated in the lung to concentrations greater than that in the plasma with the highest amount found between 3 and 12 hr. From 3 hr after dosing until 24 hr, there was a progressive increase in 14C label incorporated into spermidine, indicating that putrescine was converted to spermidine. Using autoradiographic techniques in lung slices the [3H]oligoamines were found in the alveolar epithelial type II. Clara and very probably the alveolar type I cells. With [3H]paraquat, the presence was detected only in the alveolar type II cells. Likewise, in the isolated perfused rat lung or following s.c. dosing of rats with [3H]putrescine the radiolabel was located only in the alveolar type II cell. We have suggested that the most likely explanation for the differences in localisation of label between in vitro and in vivo studies resulted from the use of [3H] label of different specific activity. Consequently we have concluded that the cell types with the ability of accumulate paraquat and oligoamines were the alveolar epithelial type I and type II cells and Clara cells.

The importance of epithelial uptake systems in lung toxicity.

The discovery that the herbicide paraquat was selectively accumulated by the lung, both in vivo and in vitro, in comparison with other tissues, provided an explanation for its selective toxicity to the lung. This uptake process is energy dependent and obeys saturation kinetics. A characterization of the process led to the identification of endogenous chemicals that are the natural substrates for the system. Among these are a series of diamines and polyamines, as well as the diaminodisulfide cystamine. It appears that paraquat, because of specific structural similarities to these endogenous polyamines, is mistakenly accumulated by the lung. This uptake process is specifically located in the alveolar Type II cell, the Clara cell, and probably the alveolar Type I cell. With the development of knowledge of the structural requirements of chemicals to be accumulated by this system, it is possible to predict which chemicals will be accumulated by the lung or design molecules that are targeted to the alveolar epithelial and Clara cells. In the wider perspective, this polyamine uptake system has been found on a number of cancerous cells or tissues. With the knowledge of the uptake system in the lung, it should be possible to design drugs that will be specifically concentrated in cells that possess this system.

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.

The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat

Abstract The role of glutathione (GSH) in the neurotoxicity produced following a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) has been investigated in rats. L-CPA-induced neurotoxicity was characterised by up to 80–90% loss in cerebellar granule cells and cerebellar oedema leading to locomotor dysfunction. Neurochemically, L-CPA-induced neurotoxicity produced a reduction in the concentration of aspartate and glutamate in the cerebellum and a reduction in the density of NMDA receptors in the cerebellar cortex, whilst there was an increase in cerebellar glycine, glutamine and GABA concentrations. Treatment of rats with buthionine sulfoximine (BSO) at 1 g/kg, i.p., an inhibitor of GSH synthesis, potentiated the toxicity of L-CPA, such that many of the neurochemical markers were significantly different from controls at earlier time points, compared to animals which had received L-CPA alone, and toxicity was also seen in the kidney of BSO plus L-CPA treated rats. In contrast, supplementing GSH concentrations by administration of the isopropyl ester of glutathione (ip-GSH) at 1 g/kg, s.c., was able to protect rats against L-CPA neurotoxicity and prevent many of the neurochemical changes. In order to assess whether the depletion of GSH in the rat cerebellum following L-CPA treatment was related to the delivery of cysteine or cystine, the accumulation of [14C] cystine into cerebellar slices was characterised and found to be energy dependent, Na+ independent and obey saturation kinetics with an apparent Km of 77 μM and an apparent Vmax of 450 nmol/g wet weight per h. The accumulation of cystine into cerebellar slices was non-competitively inhibited by the cysteine conjugate of L-CPA with an apparent Ki of approximately 60 μM, whilst glutamate only inhibited cystine accumulation at doses which were cytotoxic to cerebellar slices. Hence the depletion of GSH in the rat cerebellum, following L-CPA administration, may be due to a reduction in the delivery to the brain of cysteine or cystine, one of the components required for GSH synthesis, by the cysteine conjugate of L-CPA. Our studies show the pivotal role GSH plays in cerebellar granule cell necrosis induced by L-CPA in the rat, indicating that a marked and sustained reduction in cerebellar GSH content by L-CPA may leave granule cells vulnerable to cytotoxic free radical damage leading to cell death, possibly mediated through excitatory amino acids.

Competition for Polyamine Uptake into Rat Lung Slices by WR2721 and Analogues

The objective of these studies was to determine whether a series of structurally related radioprotective agents could act as substrates for the recently identified polyamine system in the lung. We have shown that WR2721 (S-2(3-aminopropylamino)ethyl phosphorothioate), S-2(4-aminobutylamino)ethyl phosphorothioate (S-ABEP or WR2822) and S-2(7-aminoheptylamino)ethyl phosphorothioate (S-AHEP) competitively inhibit the uptake of putrescine into rat lung slices. The ability of the radioprotectors to act as substrates for the polyamine uptake system was expressed as the Ki for each compound. The Ki values for WR2721, S-ABEP and S-AHEP in the absence of dithiothreitol were 48, 57 and 7 mumol dm-3 compared to 155, 88 and 15 mumol dm-3 in the presence of dithiothreitol, indicating that the disulphide form may have a higher affinity for the transport system. By analogy with other substrates for the polyamine uptake system we have concluded that it should be possible to target radioprotectors to the alveolar epithelial type I and II cells and the Clara cells in the lung, as they prossess this uptake system, and thus protect these cells from oxidative stress.

Glutathione depletion in the liver and brain produced by 2-chloropropionic acid: relevance to cerebellar granule cell necrosis.

Abstract  L- and D-2-chloropropionic acid (L-CPA and D-CPA) produce selective damage to granule cells of the rat cerebellum by a mechanism that is not currently understood. We have demonstrated that both L- and D-CPA produce a rapid, dose and time dependent depletion of liver non-protein sulphydryl (NP-SH) content, mainly glutathione (GSH), while in the cerebellum and forebrain, there is a slower, dose and time dependent decrease in NP-SH. Twenty-four hours after a single dose of 750 mg/kg L-CPA (a dose sufficient to produce cerebellar toxicity, but a time prior to the onset of cellular necrosis), the content of total GSH was depleted by 85% in the cerebellum and to a lesser degree in the forebrain, while no increase in oxidised glutathione was observed in either tissue. In vitro both L- and D-CPA caused a marked reduction in GSH concentration when incubated with hepatic cytosol but not hepatic microsomes or brain cytosol. The hepatic cytosolic depletion appeared to be due to a direct reaction catalysed by a theta class glutathione S-transferase. A GSH adduct of L-CPA was isolated by high pressure liquid chromatography and identified by mass spectrometry as 2-S-glutathionyl propanoic acid, confirming a direct substitution reaction. No GSH adducts were formed by cerebellar or forebrain cytosol, suggesting that the particular isoform of glutathione S-transferase catalysing the reaction may not be present in the brain. We suggest that the marked and sustained CPA-mediated GSH depletion in the granule cells of the cerebellum may render these cells more vulnerable to oxidative free radical damage.