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.

Thomas H. Shepard, M.D., pioneer in embryology and teratology.

Dr. Thomas H. Shepard died on October 3, 2016 at the age of 93. He was a major figure in the fields of teratology, embryonic and fetal pathology, and pediatrics. He was beloved by his colleagues as he was by the many students and fellows whom he taught, mentored and befriended. His contributions to teratology are extraordinary and he is greatly missed. © 2017 Wiley Periodicals, Inc.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Dermal developmental toxicity of N-phenylimide herbicides in rats.

BACKGROUND: S-53482 and S-23121 are N-phenylimide herbicides and produced embryolethality, teratogenicity (mainly ventricular septal defects and wavy ribs), and growth retardation in rats in conventional oral developmental toxicity studies. Our objective in this study was to investigate whether the compounds induce developmental toxicity via the dermal route, which is more relevant to occupational exposure, hence better addressing human health risks. METHODS: S-53482 was administered dermally to rats at 30, 100, and 300 mg/kg during organogenesis, and S-23121 was administered at 200, 400, and 800 mg/kg (the maximum applicable dose level). Fetuses were obtained by a Cesarean section and examined for external, visceral, and skeletal alterations. RESULTS: Dermal exposure of rats to S-53482 at 300 mg/kg produced patterns of developmental toxicity similar to those resulting from oral exposure. Toxicity included embryolethality, teratogenicity, and growth retardation. Dermal administration of S-23121 at 800 mg/kg resulted in an increased incidence of embryonic death and ventricular septal defect, but retarded fetal growth was not observed as it was following oral exposure to S-23121. CONCLUSIONS: Based on the results, S-53482 and S-23121 were teratogenic when administered dermally to pregnant rats as were the compounds administered orally. Thus, investigation of the mechanism and its human relevancy become more important.

Difference in developmental toxicity among structurally similar N-phenylimide herbicides in rats and rabbits.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Further evidence for the role of free radicals in the limb teratogenicity of L-NAME.

BACKGROUND: L-NAME (N(G)-nitro-(L)-arginine methyl ester), a nitric oxide synthase inhibitor, causes severe limb reduction malformations when gravid rats are treated intraperitoneally on gd-17. Hemorrhages, appearing within hours of L-NAME administration, and defects at term can be significantly reduced by co-treatment with PBN (alpha-phenyl-N-t-butylnitrone), a spin trap antioxidant. We have proposed that limb defects result from ischemia-reperfusion injury. We examine the role of xanthine oxidase and ROS formation in the limb effects of L-NAME. METHODS: Gravidas were treated with L-NAME (50 mg/kg) in the presence or absence of allopurinol, a xanthine oxidase inhibitor. Spatial patterns of limb hemorrhage were determined promptly and at term as was digit length at the latter interval. Xanthine oxidase activities were assayed in control and treated limbs with and without allopurinol co-treatment. RESULTS: Allopurinol significantly reduced hemorrhage severity in a dose-responsive fashion when fetuses were examined at term. Higher doses of allopurinol significantly preserved digit length. Xanthine oxidase activities in fetal limb were significantly increased by L-NAME treatment whereas co-treatment with allopurinol restored activities to near-control levels. CONCLUSIONS: These findings support the role of excess reactive oxygen species (ROS) formation in L-NAME-induced limb reduction. We propose that nitric oxide (NO) depletion by L-NAME interferes with vascular integrity, and causes vasoconstriction. Resultant hypoxia stimulates superoxide formation and nitric oxide formation catalyzed by the inducible isoform of nitric oxide synthase. The reduction products of superoxide or the products of its reaction with nitric oxide oxidize or nitrate endothelial components resulting in limb reduction defects.

Involvement of mitochondria and other free radical sources in normal and abnormal fetal development.

Shepard and Mackler have documented quantitative increases in mitochondrial cristae between gestational days 10 and 14 in rats accompanied by decreased glucose utilization and increased NADH oxidase activity. Findings show a shift from glycolytic to oxidative metabolism starting at around the time of implantation. Exposure to many substances that cause transient uteroplacental hypoperfusion, including cocaine, phenytoin, calcium channel blockers, and nitric oxide synthase (NOS) inhibitors, induce limb and central nervous system (CNS) malformations while sparing the heart. We have reported that isolated electron transport particles prepared from sensitive tissues show reduced NADH oxidase activities compared with insensitive heart. They also have significantly greater superoxide formation in association with significantly reduced superoxide dismutase activities. NOS inhibitors induce severe limb reductions in late gestation. Exposure is associated with hemorrhage and nitrotyrosine (NT) formation shortly after treatment. Hemorrhage, malformations, and NT formation can be significantly reduced by coadministration of PBN, allopurinol, or aminoguanidine. On the basis of these findings, we have proposed a role for the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the genesis of limb reduction defects. It is important to note that limb reduction defects occur in humans ( approximately 0.22/1000) and have been associated with the agents listed above.