Diquat Toxicity Research Articles

Effects of the new nitrosourea derivative, fotemustine, on the glutathione reductase activity in rat tissues in vivo and in isolated rat hepatocytes

Fotemustine, a new clinically active nitrosourea, is demonstrated herein to be a poor inhibitor of glutathione reductase activity from rat liver, lung and kidney cytosols. In order to show that an intracellular step of activation does not lead to a toxic intermediary metabolite, rat hepatocytes were incubated with fotemustine. Their glutathione-related pathways were checked and shown not to be altered, while under similar experimental conditions BCNU was shown to be dramatically harmful. Furthermore, association of fotemustine with a H 2O 2 production leading drug, diquat, was shown to be inefficient—while BCNU is efficient—in potentiating the diquat toxicity. Considering the role of glutathione level in the detoxification of mutagens and carcinogens, the advantage of foemustine over BCNU in therapeutic use seems substantiated.

Paraquat and diquat: mechanisms of toxicity

The authors review the mechanisms of paraquat and diquat toxicity. They discuss the generation of multiple toxic active forms of oxygen. The pulmonary concentration of paraquat seems to be due to a mechanism of active concentration, which seems to be used by polyamines and diamines in normal situations. Considerations on therapeutics attempts are made.

Effect of diquat on the distribution of iron in rat liver

Diquat toxicity is proposed to be mediated through the generation of active oxygen species; however, the exact role of active oxygen in toxicity is not known. The generation of damaging oxygen radicals requires transition metals such as iron. In vitro studies have shown that redox cycling of diquat results in the release of iron from ferritin, thus increasing the potential for active oxygen species generation. We sought to determine if diquat administration to male Sprague-Dawley rats would result in the release of iron from ferritin in vivo. Rats were treated with diquat dibromide (20 mg/kg body weight) and the effect on the iron distribution in liver was determined. The results show that diquat-treated animals had increased levels of hepatic low molecular weight chelatable iron (LMWC-Fe) and decreased levels of hepatic ferritin iron when compared to saline-treated animals. These results suggest that diquat toxicity may be associated with the release of iron from ferritin in vivo and that iron release from ferritin may be a process common to other free radical mediated toxicities.

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: Involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O ⨪ 2), hydrogen peroxide (H 2O 2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H 2O 2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of (O ⨪ 2) remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that (O ⨪ 2) is unlikely to significantly contribute to the reduction of Fe 3+ to Fe 2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H 2O 2/Fe 2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.

Application of tracer techniques to continuous-flow toxicity testing

Frequent toxicant analyses are essential for good quality data in long-term continuous-flow tests. Due to the time consuming and costly chemical analyses, exposure levels are measured at best on a daily basis. These infrequent determinations may not detect variability in toxicant concentrations that could result in test failures. To minimize repetitive testing and improve data quality, a dye tracer method was evaluated. Rhodamine WT was selected as a toxicant tracer because of easy detectability, low toxicity to aquatic organisms, and negligible transformation in the aquatic environment. Results over a 24 h period showed that rhodamine reliably predicted the toxicant (diquat) concentrations with an r value of 0.99. Based upon these data, two replicate long-term tests with and without tracer were carried out exposing fathead minnows Pimephales promelas to diquat (1:1′-ethylene-2:2′-dipiridium dibromide). The test results indicated that the added rhodamine WT did not alter the diquat toxicity to fathead minnows using LC 50 and EC 50 values for comparisons. From these findings it is concluded that dye tracers are suitable toxicant surrogates. Their use in flow-through tests allow more frequent analyses which result in better data and minimizes experimental failure.

Lung fibrosis induced by diquat after intratracheal administration.

Diquat toxicity to lung was evaluated at various intervals after intratracheal administration to rats. Twelve hours after the injection, both type I and type II pneumocytes developed degenerative changes which were similar in nature to those induced by paraquat. A much larger dose of diquat, however, was needed for the changes compared to paraquat, which is known to be actively taken up by the lung. This morphological evidence suggests that diquat may cause alveolar damage by the same mechanism as paraquat, although it is not actively taken up by the lung. Moreover, initial alveolar damage induced by diquat was followed by lung fibrosis, as with paraquat. Since paraquat is a well known fibrogenic agent, the common property of these two agents in alveolar damage may be a key to interpreting the development of lung fibrosis.

Effects of oxidative stress caused by hyperoxia and diquat. A study in isolated hepatocytes.

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated. The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyl)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below approximately 6 nmol/10(6) cells. The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant alpha-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxyl radical scavenger alpha-keto-gamma-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation. The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase - glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H2O2 and lipid hydroperoxides.

The toxic effect of diquat on the rat lung after intratracheal administration

Diquat toxicity to the lung was evaluated in rats. Diquat was administered intratracheally (i.t.) and its toxic potential was compared with that of paraquat. Diquat showed toxic effects in the lung when administered i.t. In previous research, diquat administered by the intravenous (i.v.) or oral (p.o.) route had little effect on the lung. A sufficient dose of diquat administered i.t. would be potentially toxic to the lung, but diquat is much less toxic to the lung than paraquat, which is actively taken up by lung tissue. This suggests that diquat is not actively taken up by lung tissue when administered i.t., but may be passively taken up by the lung tissue and induce lung damage.

Toxicity studies in isolated hepatocytes from selenium-deficient rats and vitamin E-deficient rats

Isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats were treated with cumene hydroperoxide (CuOOH), phorone (diisopropylene acetone), acetaminophen, and diquat. The effect of these chemicals on cell viability, glutathione synthesis and release, and lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances was determined during a 4-hr incubation in a complete medium under 95% O 2:5% CO 2 at 37°C. CuOOH-treated (0.5 m m) selenium-deficient and vitamin E-deficient hepatocytes lost viability sooner than control hepatocytes. Thus, loss of selenium or vitamin E from the hepatocyte resulted in a cell more susceptible to damage by CuOOH. Phorone treatment (1.65 m m) resulted in depletion of intracellular glutathione in all three groups to approximately 20% of that in untreated hepatocytes. Cell viability and TBA-reactive substances were the same in treated and untreated hepatocytes. Thus, lowering of intracellular glutathione did not result in the spontaneous loss of cell viability or increased lipid peroxidation in selenium-deficient or in vitamin E-deficient hepatocytes. Acetaminophen appeared to be less toxic to selenium-deficient hepatocytes than to controls. This finding is in agreement with whole animal studies reported previously showing that selenium deficiency protects rats against acetaminophen hepatotoxicity. A potential explanation of this result is stimulation of glutathione synthesis by selenium deficiency. Severely vitamin E-deficient hepatocytes were protected from cell death by 12.5 and 25.0 m m acetaminophe, apparently by its antioxidant properties, while 50.0 m m acetaminophen was toxic to them. At all concentrations used, acetaminophen decreased the TBA-reactive substances present in the hepatocyte suspensions. Diquat (0.1 m m) caused more rapid cell death and higher levels of TBA-reactive substances in selenium-deficient hepatocytes than in control hepatocytes. Diquat toxicity in selenium-deficient isolated hepatocytes was not as severe as its toxicity in selenium-deficient whole animals, however.

Modification of chemical toxicity by selenium deficiency

Selenium deficiency causes a number of hepatic metabolic alterations in the rat which could lead to changes in chemical toxicity. It causes a decrease in glutathione peroxidase activity, an increase in glutathione S-transferase activity, and an increase in the rate of glutathione synthesis. The hepatotoxicities of three compounds which bind to glutathione S-transferase; iodipamide, acetaminophen, and aflatoxin B 1, are decreased by selenium deficiency. The toxicity of redox cycling compounds is generally increased by selenium deficiency and is accompanied by evidence of lipid peroxidation. Thus, nitrofurantoin (100 mg/kg) causes renal tubular necrosis in selenium-deficient rats but not in controls. Selenium-deficient rats are much more sensitive to diquat toxicity than are controls. Lethality of diquat in selenium-deficient rats appears to be causally linked to lipid peroxidation. Lethality of diquat in control rats is not linked to lipid peroxidation. The effect of selenium does not appear to be mediated by glutathione peroxidase, however, indicating that selenium has another oxidant defense function. Another interesting observation made was that increases in inspired O 2 tension decreased ethane production (lipid peroxidation) in selenium-deficient and in control rats given diquat. Thus, O 2 appears to prevent diquat-induced lipid peroxidation.

Modification of Chemical Toxicity by Selenium Deficiency

Modification of Chemical Toxicity by Selenium Deficiency. Burk, R.F. and Lane, J.M. (1983). Fundam. Appl. Toxicol. 3: 218–221. Selenium deficiency causes a number of hepatic metabolic alterations in the rat which could lead to changes in chemical toxicity. It causes a decrease in glutathione peroxidase activity, an increase in glutathione S-transferase activity, and an increase in the rate of glutathione synthesis. The hepatotoxicities of three compounds which bind to glutathione S-transferase; iodipamide, acetaminophen, and aflatoxin B1, are decreased by selenium deficiency. The toxicity of redox cycling compounds is generally increased by selenium deficiency and is accompanied by evidence of lipid peroxidation. Thus, nitrofurantoin (100 mg/kg) causes renal tubular necrosis in selenium-deficient rats but not in controls. Selenium-deficient rats are much more sensitive to diquat toxicity than are controls. Lethality of diquat in selenium-deficient rats appears to be causally linked to lipid peroxidation. Lethality of diquat in control rats is not linked to lipid peroxidation. The effect of selenium does not appear to be mediated by glutathione peroxidase, however, indicating that selenium has another oxidant defense function. Another interesting observation made was that increases in inspired O2 tension decreased ethane production (lipid peroxidation) in selenium-deficient and in control rats given diquat. Thus, O2 appears to prevent diquat-induced lipid peroxidation.

The effect of high concentrations of oxygen on paraquat and diquat toxicity in rats.

High concentrations of oxygen are known to enhance the toxic effects of paraquat in the lung. We have examined the effects of paraquat (2.5 mg/kg or 20 mg/kg subcutaneously) and diquat (10 mg/kg or 20 mg/kg subcutaneously) on mortality and lung pathology in rats exposed to air or to an atmosphere of 85% oxygen. Our results show a 10-fold increase in mortality when paraquat is given to rats placed in 85% oxygen rather than air, but only a 2-fold increase in the lethality of diquat. Lung damage typical of early paraquat intoxication is seen following 20 mg/kg paraquat in air or oxygen, with damage to type I and type II alveolar cells. Selective damage to the type II cell is produced by lower levels of paraquat (2.5 mg/kg) and by 20 mg/kg diquat, both in 85% oxygen, other cell types showed little change. Lung damage is minimal following 2.5 mg/kg paraquat or 20 mg/kg diquat in air, or exposure to 85% oxygen alone. It is suggested that the type II cell may be the primary target cell for paraquat and diquat in the lung.

Tissue distribution of paraquat and diquat after oral administration in rats

The differential distribution of paraquat and diquat in liver, kidney, lung, brain and heart has been studied after oral administration of the LD 50 or LD 50 2 to rats. Paraquat concentrations were highest in all organs at 24 hours; at this time concentrations in kidney and lung were 2 to 3 times higher than at 2 hours. In contrast, with the exception of kidney, tissue diquat concentrations were highest at 2 hours. There was severe lung damage at 24 hours after paraquat; diquat did not produce severe lung lesions, but caused intestinal distention and diarrhea. These findings suggest that the difference in the toxicity of paraquat and diquat is related to their difference in tissue distribution and excretion.

The influence of hyperoxia on the acute toxicity of paraquat and diquat.

Hyperoxia has been shown to enhance the toxicity of the herbicide paraquat. Experiments were conducted to learn more about the effects of oxygen following acute poisoning with paraquat as well as the structurally related herbicide, diquat. Rats were injected intravenously with various doses of diquat or paraquat and placed into an atmosphere of either 100% oxygen or room air. The time required for 50% lethality (LT50) of both diquat and paraquat was greatly diminished by hyperoxia and was dependent upon the herbicide dosage. Rats treated with 40 or 80 mg/kg diquat and exposed to 100% oxygen had a shorter LT50 than those treated similarly with paraquat. A dose of 20 mg/kg was equitoxic in 100% oxygen while rats treated with 5 or 10 mg/kg diquat had a longer LT50 than rats treated with the same dose of paraquat. All animals exhibited severe respiratory distress terminally. The plasma concentrations and tissue distribution of either herbicide at 20 mg/kg were the same in oxygen and air exposed animals. When oxygen concentrations were varied between 100% and 60% rats treated with 20 mg/kg diquat or paraquat exhibited increasing but equal LT50's. In 40% oxygen diquat treated rats died more rapidly than paraquat treated rats. These data demonstrate a toxic interaction between hyperoxia and diquat as well as paraquat.

Combinations of Diquat and Several Cations for Control of Hydrilla (Hydrilla verticillata)

Numerous cations were evaluated as possible substitutes for copper in mixtures with diquat (6,7-dihydrodipyrido[1,2-α:2′,1′-c] pyrazinediium ion) for control of hydrilla [Hydrilla verticillata(L.f.) Royle]. Several cations, including ferrous and ferric iron, when substituted for equal weights of copper in the mixtures, were as effective as copper in enhancing the phytotoxicity of diquat to hydrilla. In most instances, death of the test plants occurred more rapidly when the substitutes were used. Ferric chloride, because of its apparent innocuous behavior in the aquatic environment, is a desirable substitute for the more toxic copper. Bluegill sunfish (Lepomis macrochirusRAf.) were unaffected by week-long exposure to ferric chloride at concentrations as high as 100 ppmw. Since ferric chloride alone exhibited no toxicity to hydrilla in the range of concentrations tested, the greater phytotoxicity of the mixtures probably was due to the increased toxicity of diquat. In evaluations using other species of aquatic weeds, neither the addition of copper nor the addition of other cations increased the phytotoxicity of diquat. In most tests, the mixtures of diquat with other cations reduced the level of activity of diquat on weeds other than hydrilla.

Fetal toxicity and distribution of paraquat and diquat in mice and rats

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 toxicity of diquat.

<b>Clark, D. G. and Hurst, E. W. (1970).</b><i>Brit. J. industr. Med.,</i><b>27,</b> 51-55. <b>The toxicity of diquat.</b> The acute toxicity of diquat has been assessed in several species. The oral LD₅₀ ranged from about 30 mg/kg in cattle to 231 mg/kg in the rat. Large doses of diquat gave rise to symptoms indicative of an action on the central nervous system, but smaller doses did not suggest an obvious mode of action to account for the deaths, which were sometimes delayed for up to 14 days. The 24-hour percutaneous LD₅₀ in the rabbit was greater than 400 mg/kg. This dose did not irritate the skin. A drop of a 20% aqueous solution of diquat in the conjunctival sac of the rabbit eye caused only slight irritation. The chronic administration of diquat dichloride in the diet for several months led to bilateral cataract in the rat and the dog. A concentration of 0·05% in the rat diet caused bilateral opacities in all rats within 12 months, but 0·001% diquat did not cause any opacities in two years. Bilateral opacities of the lenses of all dogs occurred within 12 months of administration of 15 mg/kg/day, but 1·7 mg/kg/day was without effect after four years.