Flumioxazin, a PPO inhibitor: A weight-of-evidence consideration of its mode of action as a developmental toxicant in the rat and its relevance to humans.

Flumioxazin, is a herbicide that has inhibitory activity on protoporphyrinogen oxidase (PPO), a key enzyme in the biosynthetic pathway for heme. Flumioxazin induces anemia and developmental toxicity in rats, including ventricular septal defect and embryofetal death. Studies to elucidate the mode of action (MOA) of flumioxazin as a developmental toxicant and to evaluate its relevance to humans have been undertaken. The MOA in the rat has now been elucidated. The first key event is PPO inhibition, which results in reduced heme synthesis in embryonic erythroblasts. The critical window for this effect is gestational day 12 when almost all erythroblasts are at the polychromatophilic stage, synthesizing heme very actively. Embryonic anemia/hypoxemia is induced and the heart pumps more strongly as a compensatory action during organogenesis, leading to thinning of the ventricular walls and failure of the interventricular septum to build completely and close. Investigations showed that this MOA is specific to rats and has no relevancy to humans. Flumioxazin inhibited PPO in rat hepatocyte mitochondria more strongly than in human. A 3-dimensional molecular simulation revealed that species differences in binding affinity of flumioxazin to PPO, observed previously in vitro, were due to differences in binding free energy. In vitro studies using several types of rat and human cells (erythroblasts derived from erythroleukemia cell lines, cord blood, or pluripotent stem cells), showed that flumioxazin decreased heme synthesis in rat cells but not in human cells, demonstrating a clear, qualitative species difference. Considering all available information, including data from PBPK modelling in rat and human, as well as the fact that anemia is not a symptom in patients with variegate porphyria, a congenital hereditary PPO defect, shows that the sequence of events leading to adverse effects in the rat embryo and fetus are very unlikely to occur in humans.

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.

An Atypical Acidophil Cell Line Tumor Showing Focal Differentiation Toward Both Growth Hormone and Prolactin Cells.

We report a case of giant pituitary adenoma in a child. Computerized tomography (CT) scan revealed a suprasellar extension tumor mass with hydrocephalus. There was no clinical evidence of acromegaly, gigantism, and other hormonal symptoms. Endocrinologic studies showed within normal value of serum growth hormone (GH: 4.2 ng/mL) and slightly increased levels of prolactin (PRL: 78 ng/mL) and other pituitary hormone values were within normal range. On suppression test by bromocryptin, both GH and PRL levels were reduced. Histopathological findings revealed that the tumor consisted of predominantly chromophobic and partly eosinophilic adenoma cells. Immunohistochemical staining detected GH and PRL in a small number of distinctly different adenoma cells, respectively. Nonradioactive in situ hybridization (ISH) also showed GH and PRL mRNA expression in identical immunopositive cells. Electron microscopy (EM) demonstrated adenoma cells with moderate or small numbers of two types of dense granules and without fibrous body which are characteristic of sparsely granulated GH-cell adenomas. The adenoma does not fit into any classification but may be an atypical acidophil cell line tumor showing focal differentiation toward both GH and PRL cells.

Mixed growth hormone cell- prolactin cell pituitary adenoma with acromegaly: α-subunit most growth hormone cells.

A 51 -year-old woman with mixed growth hormone (GH) cell-prolactin (PRL) cell pituitary adenoma is presented. She had clinical signs due to hypersecretion of GH and PRL. Resected tissue was studied immunohistochemically and morphologically. The serial sections revealed that GH and α-subunit were co-localized in most cells, while GH and PRL were localized in different cells.

Silent mixed growth hormone cell-prolactin cell pituitary adenoma.

The case of a 51 -year-old man with recurrent nonfunctioning pituitary adenoma is presented. Despite clinically and endocrinologically normal pituitary function in regard to growth hormone and prolactin, many growth hormone- and prolactin-positive cells were immunohis-tochemically detected in adenoma tissue. Furthermore, a quite rare tumor of silent mixed growth hormone cell-prolactin cell pituitary adenoma was confirmed by the double-labeling immunoelectron-microscopical study.