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.

Metabolism of metofluthrin in rats: I. Identification of metabolites.

1. Metofluthrin (2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (Z/E)-(1R)-trans-2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate) is a novel pyrethroid insecticide, which has E/Z isomers at prop-1-enyl group. 2. Rats were orally dosed with each [14C]-labelled E/Z isomer, and the excreta were collected for isolation and identification of metabolites. Analysis of the excreta by LC/MS and NMR revealed formation of 33 and 23 (total 42) metabolites from rats dosed with Z-isomer and E-isomer, respectively. 3. Major metabolic reactions were cleavage of ester linkage, O-demethylation, hydroxylation, epoxidation or reduction of double bond, glutathione conjugation and its further metabolism, hydroxylation of epoxide and formation of lactone ring. Notably, the acid side, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, was much more variously metabolised compared to chrysanthemic acid, the acid side of the known pyrethroids. 4. Major metabolites for Z-isomer mostly retained ester linkage with 1,2-dihydroxypropyl group and/or 2-methylalcohol of cyclopropane ring, while most of those for E-isomer received hydrolysis of the ester linkage without oxidation at the 1-propenyl group or the gem-methyl groups, suggesting epoxidation and hydroxylation could occur more easily on Z-isomer. 5. As the novel metabolic pathways for pyrethroids, isomerisation of ω-carboxylic acid moiety, reduction or hydration of double bond and cleavage of cyclopropane ring via epoxidation were suggested.

Metabolism of metofluthrin in rats: II. Excretion, distribution and amount of metabolites.

1. 14 C-Labelled E/Z isomers of a synthetic pyrethroid metofluthrin ((E/Z)-(1 R,3 R)-2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate, abbreviated as RTE/RTZ, respectively) were used for rat metabolism studies. 14 C-RTE or RTZ labelled at the carbonyl-carbon [acid-14C] or the methoxymethylbenzyl-α-carbon [alcohol-14 C] was administered orally to rats at 1 and 20 mg/kg. 2. Dosed compounds were mostly absorbed, metabolised, and rapidly excreted. Dose-related increase in blood AUC suggested no saturation of absorption at the high dose. Blood 14 C was maximal at 3-8 h and decreased with a half-life of 52-163 h. Radioactivity in tissues, blood and plasma decreased basically at the same rate and the sum fell below 0.2% of the dose at 168 h. 3. Although the major metabolic pathways of the isomers, that is, ester cleavage, O-demethylation and ω-oxidation, were similar, there was a notable difference. The RTZ double bond commonly undergoes epoxidation while RTE double bond mainly undergoes glutathione conjugation, which causes faster elimination from plasma and greater excretion into faeces on RTE. Faster urinary excretion and elimination from blood were observed for the alcohol moiety than the acid moiety. 4. In conclusion, this study described the overall metabolic profiles of metofluthrin and identified the differences in metabolic breakdown between the isomers. No marked sex-/dose-related differences were observed.

Biotransformation and enzymatic reactions of synthetic pyrethroids in mammals.

Synthetic pyrethroids, a major insecticide group, are used worldwide for controlling indoor and agricultural pests. Extensive mammalian metabolism studies have been carried out since the late 1960s, and major metabolic reactions have been found to be oxidation of the acid or alcohol moiety, ester cleavage, and conjugation reactions. In addition, various conjugation reactions occur in mammals, forming hydrophilic and lipophilic conjugates. Pyrethroids are generally rapidly metabolized in mammals and completely excreted from the body in a short period. Human and laboratory animals share similar metabolic reactions for pyrethroids. Oxidation reactions in humans are mediated by several CYP isoforms. On the other hand, ester bonds of pyrethroids are hydrolyzed mainly by carboxylesterase(s).

Metabolism of 2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether (pyridalyl) in rats after repeated oral administration and a simple physiologically based pharmacokinetic modeling in brown and white adipose tissues.

Male and female Sprague-Dawley rats received repeated oral administration of 14C-2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3- [5-(trifluoromethyl)-2-pyridyloxy]propyl ether (14C-pyridalyl) at 5 mg/kg/day for 14 consecutive days, and 14C excretion, 14C concentration in tissues, and the metabolic fate were determined. Most 14C was excreted into feces. The 14C concentrations in the blood and tissues attained steady-state levels at days 6 to 10, whereas those in white adipose tissues increased until day 14. Tissue 14C concentrations were highest in brown and white adipose tissue (38.37-57.50 ppm) but were 5.60 ppm or less in all the other tissues. Total 14C residues in blood and tissues on the 27th day after the first administration accounted for 2.6 to 3.2% of the total dose. A major fecal metabolite resulted from O-dealkylation. Analysis of metabolites in tissues revealed that the majority of 14C in perirenal adipose tissue and lungs was pyridalyl, accounting for greater than 90 and 60%, respectively, of the total, whereas a major metabolite in whole blood, kidneys, and liver was a dehalogenated metabolite. The experimental data were simulated with simple physiologically based pharmacokinetics using four-compartment models with assumption of lymphatic absorption and membrane permeability in adipose tissues. The different kinetics in brown and white adipose tissues was reasonably predicted in this model, with large distribution volume in adipose tissues and high hepatic clearance in liver. Sex-related difference of pyridalyl concentration in liver was considered to be a result of different unbound fraction times the hepatic intrinsic clearance (f x CL(int)) of 1.8 and 12 l/h for male and female, respectively.

Metabolism of pyridalyl in rats.

Metabolism of pyridalyl [2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether] was examined in male and female Sprague-Dawley rats. After a single oral administration of [dichlorophenyl-(14)C]pyridalyl at 5 or 500 mg/kg, the (14)C concentration in blood reached maxima at 2 to 10 h and then decreased rapidly with a biological half-life of approximately 11 to 12 h. (14)C concentrations in liver, fat, adrenal gland, and spleen were relatively high at a low dose, reaching 2.3 to 2.7, 1.9 to 2.3, 1.1 to 1.9, and 1.4 ppm, respectively, in these tissues at 2 to 24 h after administration. Although (14)C elimination from fat and hair and skin was relatively slow compared with that from other tissues, the total residue on the 7th day was low, in the range of 1.3 to 2.3% of the dose. The (14)C distribution in tissues with a high dose, as examined by whole-body autoradiography, was similar to that observed for the low dose. Results revealed that more than 88% of the dosed radiocarbon was excreted within 1 day after administration, with cumulative (14)C excretion into urine and feces 7 days after administration of 1.7 to 2.6 and 98.7 to 101.7%, respectively. One urinary and fecal major metabolite (resulting from O-dealkylation) and two minor metabolites were identified by NMR and mass spectrometry. Residual (14)C in fat was extracted, and analysis by thin-layer chromatography showed it to be due to pyridalyl itself. No marked sex-related differences were observed in (14)C elimination, (14)C distribution, and metabolites.

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

Lack of human relevance for procymidone's developmental toxicity attributable to species difference in its kinetics and metabolism.

The agricultural fungicide procymidone can cause external genitalia abnormalities in rats but not monkeys or rabbits. To investigate the relevance of developmental findings in rats to humans, we conducted in vitro plasma protein binding studies, in vitro metabolism (biotransformation) studies using liver S9 fractions and hepatocytes, and in vivo metabolism and excretion studies using chimeric mice with humanized hepatocytes. On the basis of these results, we concluded that the metabolic and excretion profiles of procymidone in humans are similar to those in monkeys and rabbits but differ from those in rats. From the findings of this and previous studies, we judge the developmental toxicity potential of procymidone to be very low in humans.

Species differences in the developmental toxicity of procymidone-Placental transfer of procymidone in pregnant rats, rabbits, and monkeys.

To clarify species differences in the developmental toxicity of procymidone (Sumilex®, a fungicide for agricultural use), placental transfer studies were conducted using 14C-labeled procymidone in pregnant rats, rabbits, and monkeys. These studies demonstrated that maternal-to-fetal transfer of the parent compound and its hydroxylated metabolite, which are both weak anti-androgenic agents, occurred more easily than that of other metabolites, with much higher absolute concentrations achieved in the fetal circulation of rats than of rabbits or monkeys. Notably, in rats, the fetal plasma concentration of the hydroxylated metabolite was higher than that of procymidone, especially after repeated oral administration of procymidone. These results suggest that the hydroxylated metabolite is the most relevant metabolite involved in teratogenic activity in rats.

Metabolism of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and its isomer, N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), in rats. 1. Identification of metabolites in feces and urine.

Rats were orally dosed with a 1:1 diastereomixture of N-[(R)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (Delaus, S-2900) and N-[(S)-1-(2,4-dichlorophenyl)ethyl]-2-cyano-3,3-dimethylbutanamide (S-2900S), both labeled with 14C, at 200 mg/kg/day for 5 consecutive days, and 16 metabolites in urine and feces were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Several of them were unique decyanated and/or cyclic compounds (lactone, imide, cyclic amide, cyclic imino ether forms). Major biotransformation reactions of the mixture of S-2900 and S-2900S in rats are proposed on the basis of the metabolites identified in this study.

Metabolism of (Z)-(1R,3R)-Profluthrin in Rats.

When [benzyl-α-(14)C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6-8 h, and decreased (half-life; 37-52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the (14)C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ω-oxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions.

Metabolism of diethofencarb (isopropyl 3,4-diethoxyphenylcarbamate) in rats: identification of metabolites in urine.

Rats were orally given a diethofencarb (isopropyl 3,4-diethoxyphenylcarbamate) labeled with (14)C, at 300 mg/kg/day, for 4 consecutive days, and 11 metabolites in urine were purified by a combination of several chromatographic techniques. The chemical structures of all isolated metabolites were identified by spectroanalyses (NMR and MS). Ten of them were newly identified forms. Five of them were S-conjugates: three mercapturic acid conjugates, one S-methyl conjugate, and one SO-methyl conjugate. The others were two phenoxyacetic acids, hydroxyacetanilide, hydroxyisopropyl carbamate, and oxazolinone derivatives. From the results, the existence of the following reactions in rats can be concluded: (1) deethylation of the 4-ethoxy group; (2) conjugation of phenols with glutathione, gamma-glutamyltranspeptidation and depeptidation of the glutathione to form cysteine conjugates, and N-acetylation of the cysteine; (3) cleavage of the C-S linkage of cysteine conjugates followed by methylation; (4) oxidation of the S-methyl group; (5) cleavage of the carbamate linkage; (6) acetylation of the resultant amino group; (7) oxidation of the acetyl group; (8) oxidation of the isopropyl group; (9) cyclization of the oxidized isopropyl carbamate group; and (10) oxidation of the 4-ethoxy group.

Comparison of cytokine profiles in bronchoalveolar lavage fluid of mice exposed to respiratory and contact sensitizers.

Respiratory sensitization to certain low molecular weight chemicals is a big concern for workers, but unfortunately there are no validated animal models to allow identification of sensitizing chemicals in the environment. In the present study, dermally sensitized and intratracheally challenged mice were used to investigate effective indicators of respiratory sensitizers. Changes in levels of total serum IgE and nine cytokines (G-CSF, IL-4, IL-5, IL-6, IL-12(p70), IL-13, IFN-γ, MCP-1 and TNF-α) in bronchoalveolar lavage fluid (BALF) were analyzed in BALB/c mice exposed to respiratory sensitizers (phthalic anhydride (PA); diphenylmethane-4,4'-diisocyanate (MDI); toluene diisocyanate (TDI); chloramine-T (CH); and piperazine (PI)) or contact sensitizers (2,4-dinitrochlorobenzene (DNCB); and oxazolone (OXA)). Non-sensitized mice were treated dermally with solvents and challenged intratracheally with the respective test chemicals as solvent controls. Increases in total serum IgE levels were observed in all treated mice, with apparent differences in cytokine profiles. PA caused statistically significant increases in Th2 cytokines, IL-4, IL-5 and IL-13, compared with the control. IL-5 was also found to be increased with CH. The other three respiratory sensitizers caused statistically significant increases in IL-13. In contrast, no change was apparent with contact sensitizers, DNCB and OXA, in these Th2 cytokines. Increases in the Th2 cytokines indicate that all five respiratory sensitizers induced immune responses in lungs. Interestingly, elevation of G-CSF levels in BALF appeared with all five respiratory sensitizers but not the two contact sensitizers. The findings suggest that G-CSF could be effective to identify respiratory sensitizers in animal models.

In vitro metabolism of trans-permethrin and its major metabolites, PBalc and PBacid, in humans.

To estimate the metabolic profile of trans-permethrin in humans, a comparison of the in vitro metabolism of trans-permethrin in humans and rats was conducted using hepatic microsomes, and cytochrome P450 and UDP-glucuronyltransferase isoforms, which catalyze the metabolism of 3-phenoxybenzyl alcohol (PBalc) and 3-phenoxybenzoic acid (PBacid), respectively. In humans and rats, the major metabolic reaction of trans-permethrin in microsomal incubations was the cleavage of ester linkage to give PBalc, followed by oxidation to 4'-OH-PBalc, 4'-OH-PBacid, and PBacid. As to 4'-hydroxylation of PBalc, several CYPs were able to catalyze the reaction, and CYP2E1 was identified as a predominant isoform. PBacid and its conjugates (glucuronide and glycine) are major urinary metabolites of trans-permethrin in mammals. PBacid is also a metabolite of several pyrethroids, and has been used as a biomarker of human exposure to pyrethroids. Our study indicated that there was no difference in glucuronyltransferase activity of PBacid between humans and rats, and that only UGT1A9 can catalyze the glucuronidation of PBacid among human UGTs. Some UGT1A9 variants are known to have poor glucuronidation activity. From these results, it was assumed that deficiency or polymorphism of UGT1A9 might affect the profile of PBacid and its conjugates in urine collected from persons exposed to trans-permethrin or other pyrethroids. These results are helpful for understanding the metabolism of trans-permethrin in humans and determining methods for quantification of target analytes for assessment of human exposure to trans-permethrin and other pyrethroids that give PBacid and its conjugates as urinary metabolites.

Metabolism of pyridalyl in rats: excretion, distribution, and biotransformation of dichloropropenyl-labeled pyridalyl.

Metabolism of pyridalyl [2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether] labeled at position 2 of the dichloropropenyl group with 14C was investigated after single oral administration to male and female rats at 5 and 500 mg/kg. Absorbed 14C was excreted into feces (68-79%), urine (8-14%), and expired air (6-10%) in all of the groups. Regarding 14C-tissue residues on the seventh day after administration, fat showed the highest levels at 0.98-2.34 ppm and 219-221 ppm with the low and high doses, respectively. 14C-Residues in other tissues accounted for 0.03-0.32 ppm at the low dose and 3-70 ppm at the high dose. The percentages of the 14C-residue in fat were 1.50-3.16% of the dose, and those of muscle and hair and skin were also relatively high, accounting for 0.49-1.20%. Total 14C-residues in the whole body were 2.95-6.80% of the dose. Fecal metabolites in male rats treated with 500 mg/kg pyridalyl were purified by a combination of chromatographic techniques, and chemical structures of 8 metabolites were identified by NMR and MS spectrometry. The biotransformation reactions in rats were proposed to be as follows: (1) epoxidation of the double bond in the dichloropropenyl group followed by hydration, dehydrochlorination, decarboxylation, and/or mercapturic acid conjugation; (2) CO2 formation after O-dealkylation of pyridalyl and its metabolites; (3) hydroxylation of C3 in the pyridyl ring; (4) O-dealkylation of the pyridyloxy and the trimethylene groups; (5) dehydrochlorination and hydration in the dichloropropenyl group.

Metabolism of flufenpyr-ethyl in rats and mice.

The metabolism of flufenpyr-ethyl [ethyl 2-chloro-5-[1,6-dihydro-5-methyl-6-oxo-4-(trifluoromethyl)pyridazin-1-yl]-4-fluorophenoxyacetate] was examined in rats and mice. [Phenyl-(14)C]flufenpyr-ethyl was administered to rats and mice as a single oral dose at a level of 500 mg/kg, and (14)C-excretion was examined. Total (14)C-recoveries within 7 days after administration were 93.2 to 97.5% (feces, 42.0 to 46.0%; and urine, 47.2 to 55.5%) in rats and 92.6 to 96.4% (feces, 26.7 to 32.7%; and urine, 59.9 to 69.7%) in mice. (14)C-Excretion into expired air was not detected in rats (expired air of mice was not analyzed). No marked species- or sex-related differences were observed in the rate of (14)C-elimination, but a relatively higher excretion into the urine of mice was observed compared to that in rats. (14)C-residues in tissue 7 days after administration were relatively high for liver, hair, skin, and kidney, but total (14)C-residues were low, below 0.2% of the dose. An ester cleaved metabolite (S-3153acid) was the major metabolite in feces and urine. Hydroxylation of the methyl group on the C5 of the pyridazine ring and ether cleavage were also observed. No sex-related differences were observed in (14)C-elimination, (14)C-distribution, and metabolite profiles, and metabolism of flufenpyr-ethyl in rats and mice was similar. In vitro metabolism of flufenpyr-ethyl was examined using stomach and intestinal contents and blood and liver S9 fractions (postmitochondrial supernatant fractions) in rats. S-3153acid was detected as a major metabolite in the presence of intestinal contents and blood and liver S9 fractions, and a small amount was also formed in the presence of stomach contents, indicating that the parent compound is rapidly metabolized by intestinal contents and blood and liver S9 fractions through ester cleavage.