Michael S. Rose

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.

Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: Possible relevance to species differences in hepatocarcinogenicity

Trichloroethylene (TRI), administered by gavage for 10 consecutive days, at doses of 500 to 1500 mg/kg body wt increased liver weight (175% of control), decreased hepatic DNA concentration (66% of control), and increased the synthesis of DNA (500% of control; as measured by [3H]dT incorporation) in B6C3F1 mice and Alderley Park mice. Similar treatment of Osborne-Mendel rats or Alderley Park rats resulted in smaller increases in liver weight (130% of control) and decreases in DNA concentration (83% of control). No effect of TRI on DNA synthesis was seen in rats. The increased DNA synthesis in the mouse was not apparently due to regenerative hyperplasia since no signs of necrosis were seen. Furthermore the increased [3H]dT incorporation probably represented semiconservative replication of DNA and not repair, since a parallel increase of mitotic figures was observed. Hence, the liver growth noted after TRI administration appears to be due to liver cell enlargement (hypertrophy) in the rat, but both hypertrophy and hyperplasia (cell proliferation) in the mouse. An important observation has been that TRI induced the peroxisomal enzyme activities, catalase, and cyanide-insensitive palmitoyl-CoA oxidation (147 and 786% of control, respectively), in mice but not in rats. Furthermore, increases in peroxisome volume density (up to 1110% of control) were observed in mice receiving TRI. These observations lead us to suggest that the species difference in hepatocarcinogenicity of TRI, seen between the rat and mouse, is possibly due to a species difference in peroxisome proliferation and cell proliferation, the peroxisome proliferation leading to increased reactive oxygen species and DNA damage, and the cell proliferation then acting to promote this lesion.

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.

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

Abstract 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.

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

Abstract 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.

The nephrotoxicity of hexachloro-1:3-butadiene in the rat: Studies of organic anion and cation transport in renal slices and the effect of monooxygenase inducers☆

Abstract Administration of a single dose of hexachloro-1:3-butadiene (HCBD) to adult rats produced renal tubular necrosis which was reversed after about 14 days. Slices of renal cortex from HCBD-treated rats were less able to accumulate the organic anion, p -aminohippurate (PAH), whereas accumulation of the organic cation, tetraethylammonium (TEA), was unaffected. The rate of efflux of both of these ions from slices of renal cortex from HCBD-treated rats was unaltered. From these data it is concluded that the renal organic anion uptake system was selectively damaged by HCBD treatment, while the organic cation uptake system was unaffected. Treatment of animals with probenecid, an inhibitor of renal organic anion transport, did not alter the nephrotoxicity. Prior administration of a number of inducers or an inhibitor (piperonyl butoxide) of hepatic and/or renal microsomal monooxygenases did not alter the susceptibility of the kidney to HCBD-induced injury. Treatment of animals with diethylmaleate, a compound which decreases tissue nonprotein sulfhydryl content, markedly potentiated the nephrotoxicity of HCBD and led to crystalline deposits in the tubular lumen, suggesting tubular occlusion. These results indicate that cytochrome P -450-mediated reactions do not appear to play a major role in this type of renal toxicity, whereas the role of nonprotein sulfhydryl depletion in the nephrotoxicity needs further investigation.

Effects of diquat on the gastrointestinal tract of rats.

After oral administration of 900 μ mol of diquat/kg (LD50) to male rats there was a rapid accumulation of water in the lumen of the gastrointestinal tract of all animals. This reached a maximum 24 hr after dosing (excess water at 24 hr, approximately 14 ml/rat). Other tissues, especially blood, were dehydrated. Twenty-four hours after dosing the degree of hemoconcentration and the amount of excess water in the gastrointestinal tract were related to the amount of diquat administered. Subcutaneous injection of 90 μ mol of diquat/kg caused a delayed increase in water content and dry weight of the gastrointestinal tract. Severe changes were limited to a group of fatally poisoned animals which died between 2 and 8 weeks following injection. These animals had significantly increased tissue water. Following oral administration of an LD50 of diquat to rats, deaths occurred over 9 days with about 50% of these occurring within the first 3 days. Rats which did not gain weight over the first 24 hr following dosing died within the first 3 days. Animals which did not gain weight 24 hr after an oral LD50 had significantly greater gastrointestinal water content and had a significantly more pronounced hemoconcentration than the animals in the remaining group which gained weight. It is concluded that diquat has an effect on water distribution within the body following oral administration and that early deaths are associated with rapid fluid loss into the gastrointestinal tract. Death following subcutaneous dosing appears to be an unrelated phenomenon.

The toxicity and renal handling of paraquatin cynomolgus monkeys

The acute intravenous and oral toxicity of single doses of paraquat dichloride was studied in the cynomolgus monkey. Renal handling and effects upon renal function were also investigated following an oral dose of [14C]paraquat. Clinical signs consisted of vomiting, anorexia and dyspnoea. By 48 h all animals showed signs of acute renal failure with oliguria, high plasma urea and SGPT levels and metabolic acidosis. Animals dosed orally showed similar, though less severe, signs to those dosed intravenously. The oral LD50 was approx. 70 mg paraquat cation/kg. Following an oral dose plasma levels peaked by 2 h, but were constant from 12 h to 24 h. Paraquat clearance was high initially and exceeded the creatinine and urea clearance, but fell off markedly after 14 h as renal failure developed. By 18 h urine production had ceased. It is concluded that acute renal failure and acute pulmonary damage are the main causes of death, with interstitial pulmonary fibrosis being a factor in animals surviving the acute phase.

A monoclonal antibody raised to rat liver cytochrome P-448 (form c) which recognises an epitope common to many other forms of cytochrome P-450

A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem.

Early effects of diquat on plasma corticosteroid concentrations in rats

Abstract Plasma corticosteroid concentrations were significantly elevated 15 min after intraperitoneal (i.p.) injection of 90 μmoles of diquat/kg body wt ( ld 50 dose) into phenobarbitone anaesthetised rats. Similar effects were noted after subcutaneous dosing but after oral administration there was a delay of approximately 1 hr before significantly elevated concentrations were observed. The magnitude of the increase after i.p. administration of diquat was dose-related up to 26 μmoles/kg body wt, as was the duration of the response. The increase in plasma corticosteroid concentration in diquat-treated rats could be prevented by pretreating animals with dexamethasone which also reduced adrenocorticotrophic hormone (ACTH) concentration in these animals. Experiments in vivo and in vitro indicated that diquat did not increase the steroidogenic response of adrenals to ACTH. It is concluded that the increased adrenal steroid synthesis observed at early times after diquat administration is caused by release of ACTH from the pituitary.