Jun Abe

Amino acid derivative reactivity assay (ADRA) is an in chemico assay for assessing the skin sensitization potential of chemicals by evaluating the reactivity of nucleophilic reagents that mimic skin proteins. N-(2-(1-Naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), used as nucleophilic reagents, are small-molecule derivatives of two different amino acids, each with a naphthalene ring attached. The rate of decrease in the amount of NAC or NAL in the reaction solution is evaluated in this assay as an indicator of the test substance's skin sensitization ability. However, the products formed between the nucleophilic reagent and the test substance, which play an important role in vivo, are not directly identified. Therefore, six highly reactive chemicals, including the proficiency substances listed in the OECD Test Guidelines─squaric acid diethyl ester, 2-methyl-2H-isothiazol-3-one (MI), p-benzoquinone, palmitoyl chloride, diphenylcyclopropenone (DPCP), and imidazolidinyl urea (IU)─were used to determine each formed product. Samples were prepared according to the standard ADRA method, and the formed products were predicted on the basis of the reaction mechanism. Excluding DPCP, the estimated structures were validated using mass spectrometry and nuclear magnetic resonance spectrometry on the synthesized samples. In this manner, the products of each nucleophile were confirmed for all examined test substances. The estimated structure products were obtained through a series of reactions initiated by the nucleophilic attack of NAC's thiol group or NAL's amino group on the test substance's electron-deficient carbonyl carbon. However, contrary to expectations, disulfide-linked-type ring-opened products were detected in the case of MI, and products with free formaldehyde in solution were detected in the case of IU. In summary, all skin sensitizers tested herein reacted with NAC and/or NAL to give products. This supports the theoretical validity of ADRA, which provides an indirect evaluation of the formed products based on a decrease in nucleophilic reagents.

A new herbicide, epyrifenacil (S-3100), inhibits protoporphyrinogen oxidase (PPO) in plants. Repeated administration of epyrifenacil in laboratory animals led to some toxicological changes related to PPO inhibition, e.g., hepatotoxicity caused by porphyrin accumulation and anemia caused by the inhibition of heme biosynthesis. In vitro studies revealed that an ester-cleaved metabolite, S-3100-CA, is predominant in mammals, exhibits PPO-inhibitory activity, and thus is the cause of epyrifenacil-induced toxicity. To assess the human risk, the effects of species differences on the dynamics (PPO inhibition) and kinetics (liver uptake) of epyrifenacil were evaluated separately. The results of in vitro assays revealed an approximately tenfold weaker inhibition of PPO by S-3100-CA in humans than in rodents and six- to thirteen-fold less hepatic uptake of S-3100-CA in humans than in mice. Finally, it was suggested that humans are less sensitive to the toxicity of epyrifenacil than are rodents, although further mechanistic research is highly anticipated.

The metabolic fate of a newly developed herbicide, epyrifenacil, (ethyl[(3-{2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]phenoxy}pyridin-2-yl)oxy]acetate, S-3100), in rats was determined using ¹⁴C-labeled epyrifenacil. When it was administered orally to rats at 1 mg/kg, around 73–74% of the dose was absorbed, metabolized, and mainly excreted into feces within 48 h. The elimination of radioactivity in plasma and tissues was rapid, suggesting that exposure of epyrifenacil and metabolites is small. Metabolite analysis revealed that epyrifenacil was rapidly ester-cleaved to M1 and then mainly excreted into bile or further metabolized. No parent was detected in plasma, tissues, and urine. Remarkably, M1 was mainly distributed in the liver (at a concentration of 70–112 times higher than in plasma at a low dose). Furthermore, a significant sex-related difference was observed in urinary excretion of M1. Considering the above observations with those in the literature, the organic anion-transporting polypeptide (OATP) likely plays a role on the active transport of M1 in the liver and kidney.

Similar to the pharmaceutical compounds, pesticides require human safety assessment for their registration and distribution; however, it is absolutely impossible to assess human safety by dosing humans with pesticides. Thus, how to appropriately evaluate the safety of pesticides in humans remains a great subject of debate. In this article, we present some examples of pesticide toxicity studies that identify species differences in toxicity and evaluate human safety by applying combinations of novel in vivo, in vitro, and in silico techniques to separately assess the key toxicodynamic (i.e., sensitivity) and/or toxicokinetic (i.e., exposure) factors. Because it is scientifically sound, the safety assessment strategy illustrated for three compounds in this article is expected to play an important role in the human safety assessment of agricultural compounds.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, because rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/β-catenin signaling and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild-type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/β-catenin signaling when compared with those of hCAR/hPXR mice, wild-type mice, and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators whereas the hCAR/hPXR mouse is not.

Phenobarbital (PB) and Di (2-ethylhexyl) phthalate (DEHP), an anti-epileptic drug and a plasticizer used in flexible polyvinylchloride formulations, respectively, are well-known typical hepatotoxicants. This study investigated the effects of PB (100 mg/kg/day) or DEHP (500 mg/kg/day) on the endocrine system in intact juvenile/peripubertal male rats exposed for 31 days beginning on postnatal day 23. Slight hormone level changes, histopathological changes in thyroid gland or induction of UDP-glucuronosyltransferase in liver were observed in both the PB and DEHP groups. One of the assumed mechanisms inducing thyroid effects is predictable to be secondary changes based on the enhancement in thyroid hormone metabolism via the induction of hepatic microsomal enzymes. No reproductive system-related changes in organ weights, histopathology, and sexual maturation were observed in both groups. Lower testosterone level was observed in the PB group. CYP2B and CYP3A, which are involved in testosterone metabolism, were induced in liver of the PB group. There was no change of 17β-hydroxysteroid dehydrogenase activity in testis of both groups. Lower testosterone level in the PB-treated male rats was attributed to an indirect, hepatotoxicity-associated effect on the reproductive system and not to direct effects on testis such as the antiandrogenic activity and the inhibition of steroidogenesis. These results did not indicate that PB or DEHP exposure affects the endocrine system directly.

A metabolite of procymidone, Hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of Hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of Hydroxylated-PCM was less, resulting in a much lower exposure. The main excretion route of Hydroxylated-PCM-glucuronide was bile (the point that Hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone’s potential for causing teratogenicity in humans must be low compared to that in rats.