James E. Gibson

A mechanism of paraquat toxicity in mice and rats

The purpose of this study was to investigate the hypothesis that paraquat toxicity results from cyclic reduction-oxidation of paraquat in vivo, with subsequent generation of superoxide radicals and initiation of lipid peroxidation. Phenobarbital pretreatment (0.1% in drinking water for 10 days) significantly increased the paraquat LD50 in mice, but only when the mice were continued on phenobarbital after paraquat administration. Phenobarbital, through its own metabolism, may be competing for electrons which might otherwise be utilized in paraquat reduction and thus decrease paraquat toxicity. Paraquat, given at 30 mg/kg ip to mice, significantly decreased liver concentrations of the water-soluble antioxidant, reduced glutathione, and lung concentrations of lipid-soluble antioxidants. The decrease in tissue antioxidants may reflect the initiation of lipid peroxidation by paraquat. Oxygen-tolerant rats (exposure to 85% oxygen for 7 days) have increased activities of pulmonary enzymes which combat lipid peroxidation. The paraquat Lt50 (median time to death) after 45 mg/kg of paraquat ip was significantly increased in oxygentolerant rats compared to room-air-exposed controls. Rats exposed to 100 ppm paraquat in the drinking water for 3 weeks had significantly elevated activities of lung glucose-6-phosphate dehydrogenase and glutathione reductase. The cross-tolerance of oxygen and paraquat and the induction by paraquat of pulmonary enzymes that supply reducing equivalents to combat oxidative damage support the proposal that paraquat may initiate lipid peroxidation in vivo.

A comparative study of paraquat intoxication in rats, guinea pigs and monkeys

Abstract The purpose of this study was to characterize and compare the toxicity, clinical syndrome, and pathology of paraquat in rats, guinea pigs, and monkeys. The LD50 of oral paraquat was 126, 22, and 50 mg/kg in the three species, respectively. Clinical signs of intoxication were anorexia, adipsia, diarrhea, hyperpnea, dyspnea, and tachycardia. All animals were hypoxic. Monkeys which received greater than 63 mg/kg of paraquat died within 1–2 days. Death was preceded by convulsive seizures. Monkeys that received paraquat dosages of 50–53 mg/kg showed signs of dyspnea prior to death while those monkeys given 30–40 mg/kg paraquat showed signs of dyspnea for 1–5 days and subsequently developed pulmonary fibrosis. Focal necrosis was noted in liver, kidney, and the gastrointestinal tract but the primary lesion occurred in the lungs of all species. Animals that died in less than 7 days showed pulmonary hemorrhage, edema, and congestion. Rats and monkeys but not guinea pigs developed interstitial fibrosis of the lung after 7–10 days. Monkeys did not have liver and kidney dysfunction. It was concluded that the paraquat-induced pulmonary fibrosis in humans could be duplicated in monkeys and rats.

Paraquat disposition in rats, guinea pigs and monkeys.

Abstract LD50 doses of 14C-labeled paraquat were administered to rats, guinea pigs and monkeys by gavage, and radioactivity was determined in excreta and tissues. Rat urine was analyzed for paraquat metabolites by thin-layer chromatography. [14C]Paraquat was absorbed from the gastrointestinal tract and reached highest serum values 0.5–1 hr after administration. Disappearance of [14C]paraquat from serum was characterized by a rapid initial decline followed by a prolonged slow decline. Tissue paraquat values were higher than serum values in rats and guinea pigs. Relative to other tissues, paraquat accumulated transiently in the lung and reached peak concentration 32 hr after administration. In rats a major portion of administered paraquat was not absorbed from the gastrointestinal tract. At 32 hr after paraquat, 52% of the administered dose remained in the gastrointestinal tract and 17 and 14% of the administered dose was excreted in the feces and urine, respectively. No radioactivity was recovered in expired air or flatus. Excretion of paraquat in urine and feces was prolonged in all species. In monkeys paraquat was measured in urine and feces 21 days after administration. Chromatography of urine from [14C]paraquat-treated rats revealed no metabolites. The primary pathologic changes induced by paraquat in the lung may be related to the transient uptake of the chemical by that organ.

Differential toxicity of monochloroacetate, monofluoroacetate and monoiodoacetate in rats

Acute lethality and mechanisms of toxicity of monochloroacetate (MCA), monofluoroacetate (MFA) and monoiodoacetate (MIA) were compared in rats. MCA, MFA and MIA were administered to rats, and the number of dead was determined. LD90 doses of the haloacetates were administered to rats, and the time of death (LT) was noted. The effect of haloacetates on in vitro [14C] acetate oxidation in liver homogenates was determined, and Hofstee plots were used to estimate reaction kinetics. MCA binding of in vivo and in vitro sulfhydryl (SH) groups was examined as a possible mechanism of toxicity. Tissue distribution of [14C] haloacetates, LD90 dose, and of [14C]MCA, LD1 dose, was determined. The 24-hr LD50 for MFA, MIA and MCA were, respectively, 5, 60 and 108 mg/kg. The LT50 for MCA, MFA and MIA were, respectively, 130, 310 and 480 min. Reaction parameters for [14C]acetate oxidation were, Vmax = 30.5 × 102dpm14CO2min−1, apparent Km = 2.4 × 10−6, m. MCA and MFA inhibited [14C]acetate oxidation: MCA apparent Ki = 9.1 × 10−7, m, and MFA apparent Ki = 11.0 × 10−7, m. MCA and MIA reduced the SH concentration in the kidney and liver but MCA did not reduce cysteine SH concentration in vitro. [14C]MCA and [14C]MIA distributed primarily to liver and kidney, but [14C]MFA had relatively higher plasma concentrations. It was concluded that MCA, MFA and MIA produce acute toxicity by dissimilar mechanisms.

Nephrotoxicity of paraquat in mice

The nephrotoxicity of paraquat was evaluated by estimating renal function in vitro and in vivo in surviving mice 24 hr after an LD50 (7 day) dose of the herbicide. Proximal tubular function was monitored in vitro by measuring accumulation of p-aminohippurate (PAH) and N-methylnicotinamide (NMN) into renal cortical slices. Disappearance of phenolsulfonphthalein (PSP) and [14C]-paraquat from plasma was used to monitor tubular function in intact animals. Glomerular function was approximated using disappearance of iothalamate from plasma. Renal cortical accumulation of PAH and NMN in vitro was not markedly altered following paraquat poisoning. In contrast, dramatic effects were measured in vivo. The rate of disappearance of both [14C]-paraquat and PSP from plasma was significantly reduced in poisoned mice. In contrast, the rate of disappearance of iothalamate was not affected by paraquat. However, the concentration of iothalamate was higher in the plasma of treated animals, suggesting that paraquat poisoning results in a smaller volume of distribution for iothalamate but fails to alter glomerular filtration. Thus, the functional nephrotoxicity due to paraquat appears to be restricted to the proximal nephron. Elimination of paraquat is primarily via the kidney and likely involves active secretion into the urine. Since secretion requires concentration within the cells of the proximal tubules, it follows that toxic concentrations of herbicide might be reached in this area of the nephron. Furthermore, it follows that, should toxic concentrations of paraquat be reached in the kidney, subsequent impairment of renal function would impede elimination of the drug, leading to more profound toxicity in organs other than the kidney.

Liver damage following paraquat in selenium-deficient and diethyl maleate-pretreated mice.

Abstract Human paraquat poisoning often includes a transient impairment of liver function before death due to pulmonary edema and fibrosis. The purpose of this study was to examine the effect of paraquat on liver function in mice fed normal lab chow, in mice fed a selenium (Se)-deficient diet for 5 weeks, and in diethyl maleate-pretreated mice (1.2 ml/kg). Liver function was determined by evaluating plasma activity of glutamic-pyruvic transaminase (SGPT), hexobarbital sleeping time, and plasma disappearance of indocyanine green (ICG). Paraquat, in doses as high as the LD50 (30 mg/kg, ip) did not alter hexobarbital sleeping time or SGPT activity in lab chow-fed mice. Selenium-deficient, paraquat-treated mice, however, had significantly elevated SGPT activity (31 units in control mice, > 1000 units in treated group), had longer hexobarbital sleeping times (from 30 min to > 180 min), and retained ICG in plasma (15-min plasma ICG concentrations of 8.4 mg/100 ml in Se-deficient paraquat-treated group vs 1.3 mg/100 ml in controls). Liver damage in Se-deficient treated mice was also observed histologically. In mice treated with paraquat, diethyl maleate pretreatment produced similar, but not as marked, effects as Se deficiency. The results suggest that paraquat alone is not hepatotoxic in mice; however, Se deficiency or diethyl maleate pretreatment may shift the organ specific toxicity of paraquat toward the liver.

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.

Postnatal toxicity of chronically administered paraquat in mice and interactions with oxygen and bromobenzene

Abstract The purpose of this study was to investigate the effect of chronic paraquat administration on developing mice and to examine the interaction of chronic paraquat exposure with 100% oxygen and the hepatotoxin bromobenzene. Paraquat was administered at 50 and 100 ppm in the drinking water to pregnant mice beginning at Day 8 of gestation, with continued exposure to the newborns up to 42 days after birth. Neither paraquat treatment altered the postnatal growth rate; however, 100 ppm but not 50 ppm paraquat significantly increased the postnatal mortality. Both 50- and 100-ppm paraquat-treated mice were sensitized to the onset of oxygen toxicity, determined by a significant reduction in the LT50 at 42 days after birth. An enhanced sensitivity to oxygen toxicity was also detectable in 100 ppm but not 50 ppm mice at Days 1 and 28 after birth. In 42-day-old mice, 50 and 100 ppm paraquat treatment also significantly reduced the LT50 after 3100 mg/kg ip (LD85) bromobenzene. These observations suggest that the toxicity of paraquat may be mediated through an interaction with oxygen and describe possible interactions that could occur with the environmental use of paraquat.

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, 10−3 m 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.

Concomitant dietary exposure to polychlorinated biphenyls and polybrominated biphenyls: tissue distribution and arylhydrocarbon hydroxylase activity in lactating rats.

Abstract Polybrominated biphenyls (PBBs) and polychlorinated biphenyls (PCBs) stimulate microsomal mixed function monooxygenases in liver and extrahepatic tissues. These compounds accumulate to high concentration in fatty tissues and are excreted into milk. Human populations that have been exposed to PBBs are also likely to have been exposed to PCBs. Therefore, to assess potential hazard of simultaneous exposure to PCBs and PBBs, it was of interest to determine the distribution of PCBs (Aroclor 1254) and PBBs (Firemaster BP6) in several tissues of lactating rats, to determine the concentrations of both agents in milk and to study the effects of these agents on hepatic and extrahepatic arylhydrocarbon hydroxylase (AHH) after concomitant dietary exposure. Sprague-Dawley rats were placed on diets containing PBBs and/or PCBs from the eighth day of pregnancy to 14 days postpartum. Treatment with the highest dose of PBBs (200 ppm) retarded both dam and pup body weight gain. Stimulation of AHH was greater after treatment with PBBs alone than PCBs alone. Extrahepatic tissue concentrations of PCBs and PBBs were similar regardless of whether these agents were administered together or alone. Liver and milk contained lower concentrations of PBBs after treatment with an equal mixture of PCBs and PBBs than when PBBs were administered alone. Milk contained higher concentrations of PCBs than PBBs indicating that this route of excretion may be more important for PCBs.