N-phenylimide Herbicide Research Articles

Lack of human relevance for rat developmental toxicity of flumioxazin is revealed by comparative heme synthesis assay using embryonic erythroid cells derived from human and rat pluripotent stem cells.

Fetal rat anemia from flumioxazin, an N-phenylimide herbicide, is caused by suppression of heme synthesis resulting from inhibition of protoporphyrinogen oxidase (PPO). A series of studies to investigate the effects of flumioxazin have revealed that developmental toxicity is caused in rats but not in rabbits, and the adverse effects are not likely to occur in humans. In this study, as a final weight-of-evidence approach for assessing the human safety of flumioxazin, we compared the toxic potential of inhibition of heme synthesis leading to anemia between human and rat embryonic erythroid cells, which were degenerated as the target of flumioxazin in the rat developmental toxicity. To obtain embryonic erythroid cells, we established respective differentiation methods for embryonic erythroid cells from both human and rat pluripotent stem cells. Derived human and rat embryonic erythroid cells were treated with flumioxazin or dihydroartemisinin (DHA), an anti-malarial drug that causes reduction of embryonic erythroid cells and leads to anemia without species differences. In the human embryonic erythroid cells, DHA inhibited cell proliferation and heme synthesis, whereas there were no effects on heme content or cell proliferation with flumioxazin. In the rat embryonic erythroid cells, however, a dose-related reduction in heme synthesis occurred with treatment of flumioxazin and of DHA. These results confirmed that flumioxazin has no effect on heme synthesis in human embryonic erythroid cells. The present data were in accordance with the results of previous studies and demonstrated that there are no concerns in humans regarding the developmental toxicity of flumioxazin observed in rats.

Different effects of an N-phenylimide herbicide on heme biosynthesis between human and rat erythroid cells

Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects and wavy ribs) were produced by S-53482, an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. The sequence of key biological events in the mode of action has been elucidated as follows: inhibition of PPO interferes with normal heme synthesis, which causes loss of blood cells leading to fetal anemia, embryolethality and the development of malformations. In this study we investigated whether the rat is a relevant model for the assessment of the human hazard of the herbicide. To study effects on heme biosynthesis, human erythroleukemia, human cord blood, and rat erythroleukemia cells were treated with the herbicide during red cell differentiation. Protoporphyrin IX, a marker of PPO inhibition, and heme were determined. We investigated whether synchronous maturation of primitive erythropoiesis, which can contribute to massive losses of embryonic blood, occurs in rats. The population of primitive erythroblasts was observed on gestational days 11 through 14. Heme production was suppressed in rat erythroid cells. In contrast, heme reduction was not seen in both human erythroid cells when PPO was inhibited. Rats underwent synchronous maturation in primitive erythropoiesis. Our results combined with epidemiological findings that patients with deficient PPO are not anemic led us to conclude that human erythroblasts are resistant to the herbicide. It is suggested that the rat would be an inappropriate model for assessing the developmental toxicity of S-53482 in humans as rats are specifically sensitive to PPO inhibition by the herbicide.

Flumioxazin metabolism in pregnant animals and cell-based protoporphyrinogen IX oxidase (PPO) inhibition assay of fetal metabolites in various animal species to elucidate the mechanism of the rat-specific developmental toxicity

Flumioxazin, an N-phenylimide herbicide, inhibits protoporphyrinogen oxidase (PPO), a key enzyme in heme biosynthesis in mammals, and causes rat-specific developmental toxicity. The mechanism has mainly been clarified, but no research has yet focused on the contribution of its metabolites. We therefore conducted in vivo metabolism studies in pregnant rats and rabbits, and found 6 major known metabolites in excreta. There was no major rat-specific metabolite. The most abundant component in rat fetuses was APF, followed by flumioxazin and 5 identified metabolites. The concentrations of flumioxazin and these metabolites in fetuses were lower in rabbits than in rats. In vitro PPO inhibition assays with rat and human liver mitochondria showed that flumioxazin is a more potent PPO inhibitor than the metabolites. There were no species differences in relative intensity of PPO inhibition among flumioxazin and these metabolites. Based on the results of these in vivo and in vitro experiments, we concluded that flumioxazin is the causal substance of the rat-specific developmental toxicity. As a more reliable test system for research on in vitro PPO inhibition, cell-based assays with rat, rabbit, monkey, and human hepatocytes were performed. The results were consistent with those of the mitochondrial assays, and rats were more sensitive to PPO inhibition by flumioxazin than humans, while rabbits and monkeys were almost insensitive. From these results, the species difference in the developmental toxicity was concluded to be due to the difference in sensitivity of PPO to flumioxazin, and rats were confirmed to be the most sensitive of these species.

Molecular dynamics mechanism to generate species differences in inhibition of protoporphyrinogen oxidase by flumioxazin

Flumioxazin is an N-phenylimide herbicide and shows a remarkable species difference in developmental toxicity between rats and rabbits. The species difference is corresponded well to the inhibitory potency on protoporphyrinogen oxidase (PPO), an enzyme involved in chlorophyll and heme biosynthesis. In vitro experiments have shown that rat PPO is more sensitive to flumioxazin than human PPO and rabbit PPO. However, it remains unknown how the large difference in sensitivity to flumioxazin is generated in PPOs with highly conserved amino acid sequences. In order to determine the molecular dynamics (MD) mechanism responsible for these species differences, we performed MD simulations of human, rat, and rabbit PPO–flumioxazin complexes and found that rat PPO exhibited a higher binding affinity of flumioxazin than human and rabbit PPOs. A sophisticated comparative analysis of MD trajectories demonstrated that differences in the dynamics of the 107–120 loop region derived from amino acid sequence variants generated differences in the PPO–flumioxazin interactions via changes in the dynamics of Arg97. Moreover, a change in the shape of the flumioxazin-binding pocket in human PPO weakened the van der Waals forces contributing to the human PPO–flumioxazin interaction. Additionally, the positional and orientational shift in flumioxazin weakened the Coulomb force contributing to the rabbit PPO–flumioxazin interaction. These findings support the involvement of an MD mechanism in species differences in flumioxazin sensitivity.

Mechanism of Developmental Effects in Rats Caused by an N-Phenylimide Herbicide: Transient Fetal Anemia and Sequelae during Mid-to-Late Gestation.

Rat developmental toxicity including embryolethality and teratogenicity (mainly ventricular septal defects [VSDs] and wavy ribs) was produced by an N-phenylimide herbicide that inhibits protoporphyrinogen oxidase (PPO) common to chlorophyll and heme biosynthesis. Major characteristics of the developmental toxicity included species difference between rats and rabbits, compound-specific difference among structurally similar herbicides, and sensitive period. Protoporphyrin accumulation in treated fetuses closely correlated with the major characteristics. Iron deposits in erythroblastic mitochondria and degeneration of erythroblasts were observed in treated rat fetuses. In this study we investigated fetal anemia and subsequent developmental effects in rats, and inhibition of PPO in rats, rabbits, and humans by the herbicides in vitro. Fetuses were treated on gestational day (GD) 12 and removed on GDs 13 through 20. All litters were examined externally. One half of litters were examined for blood and skeletal development, and the other half for interventricular foramen closure. Effects on PPO were determined in mitochondria from embryos and adult livers. Fetal anemia in rats was evident on GDs 13 through 16. Subsequently, enlarged heart, delayed closure of the foramen, reduced serum protein, and retarded rib ossification were observed. In vitro PPO inhibition exhibited species- and compound-specific differences corresponding to the developmental toxicity. We propose that developmental toxicity results from PPO inhibition in primitive erythroblasts, causing transient fetal anemia followed by death. Compensatory enlargement of the fetal heart results in failure of interventricular foramen closure and VSD. Reduced serum protein leads to delayed ossification and wavy ribs.

Close link between protoporphyrin IX accumulation and developmental toxicity induced by N-phenylimide herbicides in rats.

S-53482, 7-fluoro-6-[(3,4,5,6-tetrahydro)phthalimido]-4-(2-propynyl)-1,4-benzoxazin-3(2H)-one (flumioxazin), is an N-phenylimide herbicide and developmentally toxic to rats, but not to rabbits. The day of greatest sensitivity to S-53482 is gestational day (GD) 12 in rats. There is a compound-specific difference in developmental toxicity among structurally similar compounds including S-23121 (N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide; teratogenic) and S-23031 (pentyl 2-chloro-4-fluoro-5-(3,4,5,6-tetrahydrophthalimido)phenoxyacetate (flumiclorac pentyl); nonteratogenic). The herbicidal action is due to photodynamic action of accumulating protoporphyrin IX (PPIX), resulting from the inhibition of protoporphyrinogen oxidase (PPO), an enzyme in porphyrin biosynthesis. Species difference in PPIX accumulation in embryos corresponded to those of the developmental toxicity. Our objective in this study was to further investigate a link between PPIX accumulation resulting from PPO inhibition and developmental toxicity. This article is part of a series of studies to be published serially. To investigate compound-specific differences, each compound was orally administered to rats on GD 12. To define peak period of PPIX accumulation, single oral treatments of S-53482 were given to rats or rabbits at 19:30 on GD 10 through GD 15. PPIX was extracted from embryos 14 hr after treatment. Remarkable PPIX accumulation was observed when treated with S-53482 or S-23121, but not with S-23031. The greatest accumulation of PPIX was observed when treated with S-53482 at 19:30 on GD 11 or GD 12. No PPIX accumulation was found on any GDs in rabbits. The developmentally toxic compounds caused PPIX accumulation in embryos. The peak period of PPIX accumulation corresponded to that of developmental effects. This correlation suggests a close link between PPO inhibition and developmental abnormality.

Difference in Developmental Toxicity Among Structurally Similar N‐Phenylimide Herbicides in Rats and Rabbits

S-53482 is an N-phenylimide herbicide and shows a remarkable species difference in developmental toxicity between rats and rabbits. The herbicide produced embryolethality, teratogenicity (mainly ventricular septal defects and wavy ribs), and growth retardation in rats, but not in rabbits. Our objective in this study was to investigate differences in developmental toxicity among N-phenylimide compounds structurally similar to S-53482 to better characterize the developmental effects of S-53482 on rat and rabbit embryos as part of research investigation to elucidate a mechanism of rat developmental toxicity produced by S-53482. This paper is part of a series of studies to be published serially. S-23121 or S-23031, both of which are structurally similar to S-53482, was orally administered to rats and rabbits during fetal organogenesis. Fetuses were obtained by cesarian section and examined for external, visceral, and skeletal alterations. S-23121 produced the similar pattern of developmental toxicity such as embryolethality, teratogenicity, and growth retardation in rats. In contrast, S-23031 showed no developmental toxicity at 1500 mg/kg. Rabbits were insensitive to all study compounds. The difference in developmental toxicity in rats was striking among N-phenylimide herbicides. The mechanism of action of developmental toxicity by S-53482 should account for the compound-specific difference as well as species difference between rats and rabbits.

Hydrolysis of N-Phenylimide Herbicide Flumioxazin and Its Anilic Acid Derivative in Aqueous Solutions

Hydrolysis of the N-phenylimide herbicide flumioxazin was studied at pH 2.5-9.0 and 25±1°C by directly analyzing a buffered aqueous solution with column-switching high performance liquid chromatography. Flumioxazin was hydrolyzed via a base-catalyzed opening of the cyclic imide moiety with half-lives of 4.1 days, 16.1 hr and 17.5 min at pH 5.0, 7.0 and 9.0, respectively. The primary hydrolysis product was the anilic acid derivative which was subsequently degraded to the corresponding aniline and dicarboxylic acid via cleavage of the amide linkage under acidic and neutral conditions, along with a partial recyclization to flumioxazin. Kinetic analysis of the pH dependency of each reaction path showed that the acid-catalyzed cleavage of the amide bond in the anilic acid was an intramolecular reaction where the undissociated form of the carboxyl group participated.

Histological changes in rat embryonic blood cells as a possible mechanism for ventricular septal defects produced by an N-phenylimide herbicide.

An N-phenylimide herbicide, S-53482, inhibits protoporphyrinogen oxidase, an enzyme common to chlorophyll and heme biosynthesis, and produces embryolethality, teratogenicity [mainly ventricular septal defects (VSD) and wavy ribs], and growth retardation in rats. In order to elucidate the mechanism of the developmental toxicity, in particular VSD, effects of the herbicide on rat embryonic blood cells were investigated histologically at the light and electron microscopic levels at 6, 12, 24, 36, and 48 h after oral administration of the chemical to pregnant rats on day 12 of gestation, the most sensitive day for toxicity. Electron and light microscopy demonstrated mitochondrial lesions, including abnormal iron deposits that were probably due to inhibition of heme biosynthesis, in erythroblasts derived from the yolk sac. Subsequently, degeneration of these erythroblasts occurred followed by erythrophagocytosis. Histologically hearts from exposed embryos had a thin ventricular wall, which may reflect a compensatory reaction to a loss of embryonic blood cells. Thus, the herbicide may induce VSD due to hematological dysfunction caused by the inhibition of heme biosynthesis rather than by direct injurious effects on the heart.

Diphenylether-like physiological and biochemical actions of S-23142, a novel N-phenyl imide herbicide

Several physiological and biochemical actions of a new experimental herbicide, S-23142 [ N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide], have been investigated. S-23142 was active under the presence of light and oxygen. Photosynthetic CO 2 fixation in soybean began to decrease 4–5 hr after the foliar treatment of S-23142, being accompanied by the appearance of visible bleaching and wilting of the plants. A large amount of ethane, the products of peroxidation of unsaturated fatty acids, was produced from the cotyledon discs of cucumber ( Cucumis sativus L.) treated with S-23142. Leakage of ATP was also observed. S-23142 did not inhibit photosynthetic oxygen evolution of the discs just after the application; however, the oxygen evolution rate decreased as the treated discs produced ethane. The results suggest that cell membrane and chloroplast membrane were deteriorated by the membrane lipid peroxidation. S-23142 also induced ethylene production and a high level of phenylalanine-ammonia lyase activity in cucumber cotyledon, which was regarded as the phenomena of stress response. Only the ethylene production was inhibited by aminoethoxyvinylglycine and cycloheximide, while the ethane production was not affected. All of these actions of S-23142 were essentially the same as those of acifluorfen ethyl except that the activity of S-23142 was more than 10 times higher than that of acifluorfen ethyl. These data strongly suggest that S-23142 belongs to the same group as diphenylethers in its mechanisms of action despite the difference in chemical structure.