Patricia E. Levi

Olfactory toxicity of methimazole: dose-response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa.

Methimazole is a compound administered to humans for the treatment of hyperthyroidism and is used experimentally as a model substrate for the flavin-containing monooxygenase (FMO) system. Previous results from this laboratory demonstrated that methimazole is an olfactory system toxicant, causing nearly complete destruction of the olfactory epithelium in the male Long-Evans rat following a single ip dose of 300 mg/kg. The present studies were undertaken to determine the dose-response relationship for methimazole-induced olfactory mucosal damage and to determine whether or not similar damage occurs as a result of oral administration, mimicking the relevant route of human exposure. We also investigated the mechanism of olfactory toxicity of methimazole by means of a structure-activity study and began the characterization of the form(s) of FMO present in the olfactory mucosa of the male Long-Evans rat. Dose-response analysis demonstrated that methimazole causes olfactory mucosal damage at doses of 25 mg/kg ip and greater. The results of gavage studies showed that a single oral dose of 50 mg/kg also caused olfactory mucosal damage. Two structurally related compounds, methylimidazole and methylpyrrole, were not olfactory toxicants, suggesting that a reactive intermediate generated in the course of metabolizing methimazole to an S-oxide is the olfactory toxic species. Microsomal incubation studies revealed the presence ofmethimazole S-oxidation activity in olfactory mucosal microsomes at levels comparable to those in liver. An anti-mouse liver FMO antibody reacted on Western blots with olfactory mucosal microsomes. These findings demonstrate a dose-response for the olfactory toxicity of methimazole and suggest that characterization of human olfactory mucosal FMO activity may be necessary to assess the potential for human risk associated with therapeutic exposure to methimazole.

Differences in oxidative dearylation and desulfuration of fenitrothion by cytochrome P-450 isozymes and in the subsequent inhibition of monooxygenase activity

Abstract The organophosphorus insecticide, fenitrothion ( O,O -dimethyl O -(3-methyl-4-nitrophenyl) phosphorothioate), a substrate for the cytochrome P -450 ( P -450) monooxygenase system, yields the corresponding phosphate triester (fenitroxon) by oxidative desulfuration (an activation reaction) and 4-nitro- m -cresol by oxidative dearylation (a detoxication reaction). Four constitutive P -450 isozymes and the major isozymes induced by phenobarbital (PB) and β-naphthoflavone (BNF) were incubated with [ 14 C]fenitrothion in reconstituted monooxygenase systems. Both metabolites were formed with all isozymes, with the ratio of oxon to cresol being characteristic of the individual P -450s. Cytochrome P -450 B2 and P -450 PB formed the highest percentage of oxon, 75 and 82%, respectively; while P -450 A1, B3, and BNF produced 55 to 60% oxon. By contrast, P -450 A2 formed the least amount of oxon, 38%, with 62% of the product being 4-nitro- m -cresol. Microsomes prepared from mice treated in vivo with organophosphates, such as fenitrothion, have reduced levels of cytochrome P -450 and associated monooxygenase activities. Presumably, this reduction is due to destruction of P -450 caused by the release of active sulfur occurring during desulfuration. To determine if some P -450 isozymes were preferentially inhibited by fenitrothion, purified isozymes were incubated with fenitrothion and subsequent p -nitroanisole O -demethylase activity was determined. Inhibition studies using purified P -450 isozymes indicated close to 100% inhibition by fenitrothion of P -450 PB, while enzyme activity of P -450 BNF and P -450 A2 was inhibited 85–90%.

Regulation of cytochrome P-450 isozymes by methylenedioxyphenyl compounds

Regulation of cytochrome P-450 isozymes 1a-1, 1a-2, and 2b-10 by methylenedioxyphenyl compounds was studied by measuring levels of mRNA, protein, and enzyme activity in hepatic tissue from C57BL/6 (Ah+) and DBA/2 (Ah-) mice dosed with isosafrole (ISO) or piperonyl butoxide (PBO). Increases in 1a-2 and 2b-10 protein were observed for ISO and PBO in both strains of mice, suggesting an Ah receptor-independent mechanism for induction of these isozymes; 1a-1 induction, however, was seen only in C57 mice. Piperonyl butoxide was the more potent inducing agent in both strains. In C57 mice treated with five dose levels of PBO, induction of 1a-1 mRNA, protein, and enzyme activity were seen at doses equal to or greater than 104 mg/kg, but were not detected at lower doses. With isosafrole, induction of 1a-1 mRNA was observed only at the highest dose tested (400 mg/kg); however, neither 1a-1 protein nor increased enzymatic activity was seen at this dose. Dose-response studies showed maximum inducible levels for 1a-2 and 2b-10 protein, beyond which the mRNAs continued to increase while the protein levels remained constant.

Pesticide-metabolizing enzymes.

Pesticides are known to function as substrates, inhibitors and inducers of drug-metabolizing enzymes, with the same compound frequently acting in more than one of these roles. Current studies of phase I metabolism of pesticides include cytochrome P450 (P450) and the flavin-containing monooxygenase (FMO), with particular reference to individual isozymes. In mouse liver, the level of FMO1 is gender dependent, FMO3 is gender specific, while FMO5 appears to be gender independent. The isozyme specificity of methylenedioxyphenyl synergists for induction of P450 in mouse liver involves P450s 1A1, 1A2 and 2B10, including a non-Ah receptor-dependent mechanism for 1A2 induction. The substrate specificity of mouse and human P450 and FMO isozymes is discussed.

Interactions of Piperonyl Butoxide with Cytochrome P450

Publisher Summary This chapter discusses interactions of piperonyl butoxide (PBO) with cytochrome P450. Cytochrome P450s occur both as constitutive and inducible enzymes, and a number of foreign compounds are known to induce specific P450 isozymes. P450 substrates serve as inducing agents, inducing the isozymes active in their metabolism. Because of the importance of the P450 enzymes in metabolism and significant health-related consequences associated with inducing or repressing specific P450 isozymes, there is considerable interest in studying P450 regulation, the activities of specific isozymes, and the chemicals involved in the interactions with P450. PBO is a substrate, an inhibitor, and an inducer of P450 in mammals and in insects. PBO could function as a P450 inducer and because of the relatively fast rate of P450 inhibition in comparison with P450 induction, the time course of effects in mammals is biphasic. PBO is not only an effective insecticide synergist because of its interaction with P450, but owing to the same interaction, it is also useful as a diagnostic tool in pesticide toxicology in the determination of whether or not P450-dependent mono-oxygenation is involved in the metabolism of a particular pesticide and whether or not P450 mono-oxygenation is involved in resistance when resistant populations are discovered.

Relative contributions of the cytochrome P450 and flavin-containing monooxygenases to the microsomal oxidation of phorate following treatment of mice with phenobarbital, hydrocortisone, acetone, and piperonyl butoxide.

Abstract The relative contributions of the cytochrome P450-dependent monooxygenase system (P450) and the flavin-containing monooxygenase (FMO) in the microsomal oxidation of phorate, O,O -diethyl S -[(ethylthio)methyl]phosphorodithioate, were investigated in mice following treatment with phenobarbital (PB), piperonyl butoxide (PBO), acetone, or hydrocortisone. PBO treatment produced a distinct biphasic effect (initial inhibition and subsequent induction) on several hepatic enzyme activities, including phorate sulfoxidation. The relative contribution by P450 to phorate sulfoxidation decreased from 76% in control to 58% at 2 hr after treatment and increased to 89% at 12 hr. Hydrocortisone treatment caused an increase in FMO activity in liver microsomes, but not in lung microsomes. Administration of acetone caused an increase in benzphetamine N -demethylation and p -nitrophenol hydroxylation in the liver, but no change in phorate sulfoxidase activity was observed, nor was the relative contributions due to P450 and FMO altered. Treatment with phenobarbital produced a large increase in both benzphetamine N -demethylase and phorate sulfoxidase activity in liver microsomes, with the percentage of phorate sulfoxidase activity due to P450 being increased from 76% in the control livers to 85% in the treated livers.

Hepatic and extrahepatic microsomal oxidation of phorate by the cytochrome P-450 and FAD-containing monooxygenase systems in the mouse

The relative contribution of the cytochrome P-450-dependent monooxygenase system (P-450) and the FAD-containing monooxygenase (FMO) in the oxidation of phorate (O,O-diethyl S-((ethylthio)methyl)phosphorodithioate) was investigated in liver, lung, and kidney microsomes isolated from male and female Dub:ICR mice. Selective inhibition was used to define the role of each enzyme system independent of the other system. Liver microsomes exhibited the greatest sulfoxidase activity, with P-450 responsible for greater than 75% of the activity. Although overall rates of sulfoxidation were much lower in lung and kidney microsomes, the FMO activity, relative to P-450, was greater. In all three organs, FMO activity appeared higher in microsomes isolated from female mice than microsomes from male mice. Phorate sulfoxide was the only metabolite detected during the linear range of the time course incubations. Extended time incubations yielded metabolites in addition to phorate sulfoxide in liver microsomes (phorate sulfone and phorate oxon sulfoxide) and lung microsomes (phorate oxon and phorate oxon sulfoxide), but not in kidney microsomes.

Oxidation of pesticides by purified cytochrome P-450 isozymes from mouse liver

5 cytochrome P-450 isozymes were purified from the livers of uninduced mice and reconstituted with purified NADPH cytochrome P-450 reductase and phospholipid. The pesticides parathion, fonofos, DEF, Mocap and profenofos were oxidized by the reconstituted monooxygenase system to form acetylcholinesterase (AChE) inhibitors. The bioactivation varied with the pesticide substrate and the cytochrome P-450 isozyme. Aldrin epoxidation occurred with all 5 isozymes, with cytochrome P-450 A1 being the most active. All fraction metabolized the pesticide synergist piperonyl butoxide (PBO) to form an inhibitory cytochrome P-450-PBO-metabolite complex. The reduced complex produced a spectrum in the Soret region which was characteristic for each of the cytochrome P-450 isozymes. Inhibition of aldrin epoxidation by PBO was found to be unrelated to the nature of the Soret spectrum.

PARATHION METABOLISM DURING PERCUTANEOUS ABSORPTION IN PERFUSED PORCINE SKIN

The metabolism and disposition of topical parathion was examined in the isolated perfused porcine skin flap (IPPSF), a novel organ perfusion method developed for in vitro studies of the pharmacology and toxicology of skin. Ethanol solutions of 14C-radiolabeled parathion (1.0 mg/ml, 0.05 mCi/ml) were applied to the surface at a dose of 40 μg/cm2 on skin flaps representing three treatment groups: control (N = 5), occluded (N = 4), and 1-aminobenzotriazole (ABT)-pretreated (N = 2). Radiolabel uptake in the perfusion medium from 0–8 hr postapplication indicated that total chemical flux and peak rates of absorption in occluded preparations were 59% and 47% lower than in the controls (P 75% lower in the ABT-pretreated IPPSFs. Thin-layer chromatographs of ethyl acetate extracts demonstrated that most of the absorbed radiolabel recovered in the perfusion medium of the controls was paraoxon (67.8 ± 2.3%, mean ± SE), with a lesser amount of p-nitrophenol (14.5 ± 5.2%) and unmetabolized parathion (16.9 ± 4.1%). Occlusion of the application site increased the fraction of p-nitrophenol in the perfusion medium (43.0 ± 7.8%, P < 0.05), at the apparent expense of paraoxon (39.6 ± 17.8%, P < 0.05), without altering the mean percentage of parent compound recovered (17.4 + 10.1%). Pretreatment of the IPPSF by addition of 50 μg ABT/g wet tissue weight into the perfusion medium blocked most of the paraoxon formation (6.6%), but not that of p-nitrophenol (11.9%), while allowing 78.5% of the parathion absorbed to penetrate intact. Total flux assessments for parathion plus its metabolites (total radiolabel absorption) in vitro varied in conjunction with its fraction metabolized within the skin. Net permeation of unchanged parathion was similar in the control (580 ng/cm2) and ABT-pretreated skin flaps (660 ng/cm2), suggesting that the diffusivity of parathion molecules through skin was not altered by this metabolic inhibitor. In contrast, occlusion reduced the net flux of intact parathion molecules to approximately 240 ng/cm2. These findings show that parathion undergoes significant biotransformation following topical application to porcine skin and that the resultant cutaneous metabolite profiles can be altered by both physical (occlusion) and chemical (ABT) means.

Regulation of cytochrome P-450 isozymes CYP1A1, CYP1A2 and CYP2B10 by three benzodioxole compounds

Three benzodioxole (BD) compounds were used to investigate the structural requirement for regulation of the cytochrome P450 isozymes, CYP1A1, CYP1A2 and CYP2B10, in mouse liver. Male mice (C57BL/6) were treated intraperitoneally for 3 days with 5-t-butyl-1,3-benzodioxole (t-BBD), 5-n-butyl-1,3-benzodioxole (n-BBD) and 5-(3-oxobutyl)-1,3-benzodioxole (o-BBD). t-BBD-induced liver microsomes showed the highest pentoxyresorufin O-dealkylation (PROD) activity, while o-BBD induced microsomes showed slightly higher activity in ethoxyresorufin O-deethylation (EROD), benzo[a]pyrene hydroxylation (BaP-OH) and acetanilide hydroxylation (Acet-OH) assays. In vitro enzyme inhibition assays showed that n-BBD inhibited EROD and Acet-OH activities more than either o-BBD or t-BBD, while PROD activity was evenly inhibited by all three compounds. Western and northern blots showed that CYP1A1 was not detectably induced by any of the three BD compounds. The levels of CYP1A2 protein and mRNA were increased in all three treated livers. In addition to CYP1A2 induction, t-BBD also induced the protein and mRNA for CYP2B10.