Ernest Hodgson

Biochemical characteristics of insect microsomes. N- and O-demethylation.

The N-demethylation of N,N-dimethyl-p-nitrophenol carbamate and the O-demethylation of p-nitroanisole by the microsomes from housefly abdomens were studied. Demethylation activity of male flies rises to a maximum at 4 days after emergence, while the maximum activity of females is not realized until 7–9 days after emergence. Homogenization with a teflon pestle is more effective than a blender, and 0.20 M potassium phosphate buffer (pH 7.8) was found to be superior to all other media tested for the preparation of the microsomes. It was apparent that the ionic strength of the preparative medium is more important than either molarity or pH for obtaining microsomes of maximum N- and O-demethylating activity. During the subsequent incubation, 0.5 to 1.0% bovine serum albumin is required for maximum activity. The pH optimum for both O- and N-demethylation is between 7.7 and 8.1 with added NADPH or with the generating system, and both reactions show the greatest activity at 30–33°. The apparent Km for pNA is 9.0 ± 0.4 × 10−5 M and that for DpNC is 4.0 ± 1.6 × 10−4 M.

A cytochrome P-450-piperonyl butoxide spectrum similar to that produced by ethyl isocyanide

Abstract Piperonyl butoxide produces a typical type I substrate difference spectrum with cytochrome P-450 in the oxidized state. However, the inclusion of NADPH with piperonyl butoxide effects a spectrum similar to that of ethyl isocyanide. This resemblance includes the location and shape of the two Soret peaks and the effect of pH on their relative magnitudes.

In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos.

Carbaryl is a widely used anticholinesterase carbamate insecticide. Although previous studies have demonstrated that carbaryl can be metabolized by cytochrome P450 (CYP), the identification and characterization of CYP isoforms involved in metabolism have not been described either in humans or in experimental animals. The in vitro metabolic activities of human liver microsomes (HLM) and human cytochrome P450 (CYP) isoforms toward carbaryl were investigated in this study. The three major metabolites, i.e. 5-hydroxycarbaryl, 4-hydroxycarbaryl and carbaryl methylol, were identified after incubation of carbaryl with HLM or individual CYP isoforms and analysis by HPLC. Most of the 16 human CYP isoforms studied showed some metabolic activity toward carbaryl. CYP1A1 and 1A2 had the greatest ability to form 5-hydroxycarbaryl, while CYP3A4 and CYP1A1 were the most active in generation of 4-hydroxycarbaryl. The production of carbaryl methylol was primarily the result of metabolism by CYP2B6. Differential activities toward carbaryl were observed among five selected individual HLM samples with the largest difference occurring in the production of carbaryl methylol. Co-incubations of carbaryl and chlorpyrifos in HLM greatly inhibited carbaryl metabolism. The ability of HLM to metabolize carbaryl was also reduced by pre-incubation of HLM with chlorpyrifos. Chlorpyrifos inhibited the generation of carbaryl methylol, catalyzed predominately by CYP2B6, more than other pathways, correlating with an earlier observation that chlorpyrifos is metabolized to its oxon primarily by CYP2B6. Therefore, carbaryl metabolism in humans and its interaction with other chemicals is reflected by the concentration of CYP isoforms in HLM and their activities in the metabolic pathways for carbaryl. (Supported by NCDA Environmental Trust Fund)

Identification of distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the mouse and rabbit

The flavin-containing monooxygenase has been purified from mouse and rabbit lung microsomes and shown to be distinct from the flavin-containing monooxygenase found in the liver of the same species. The mouse and rabbit lung monooxygenases have a unique ability to N-oxidize the primary aliphatic amine, n-octylamine, commonly included in microsomal incubations to inhibit cytochrome P-450. In the mouse lung, this compound not only serves as a substrate but is also a positive effector of metabolism. The mouse and rabbit lung enzymes have unusual pH optimum, near 9.8, compared to the liver enzymes which have peaks near pH 8.8. Using antibodies raised in goats, Ouchterlony immunodiffusion analysis indicates that the liver and lung proteins are immunochemically dissimilar.

Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes.

Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methyl-phenyl)-acetamide], alachlor [N-(methoxymethyl)-2-chloro-N-(2, 6-diethyl-phenyl)acetamide], butachlor [N-(butoxymethyl)-2-chloro-N-(2,6-diethyl-phenyl)acetamide], and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] are pre-emergent herbicides used in the production of agricultural crops. These herbicides are carcinogenic in rats: acetochlor and alachlor cause tumors in the nasal turbinates, butachlor causes stomach tumors, and metolachlor causes liver tumors. It has been suggested that the carcinogenicity of these compounds involves a complex metabolic activation pathway leading to a DNA-reactive dialkylbenzoquinone imine. Important intermediates in this pathway are 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) produced from alachlor and butachlor and 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA) produced from acetochlor and metolachlor. Subsequent metabolism of CDEPA and CMEPA produces 2,6-diethylaniline (DEA) and 2-methyl-6-ethylaniline (MEA), which are bioactivated through para-hydroxylation and subsequent oxidation to the proposed carcinogenic product dialkylbenzoquinone imine. The current study extends our earlier studies with alachlor and demonstrates that rat liver microsomes metabolize acetochlor and metolachlor to CMEPA (0.065 nmol/min/mg and 0.0133 nmol/min/mg, respectively), whereas human liver microsomes can metabolize only acetochlor to CMEPA (0.023 nmol/min/mg). Butachlor is metabolized to CDEPA to a much greater extent by rat liver microsomes (0.045 nmol/min/mg) than by human liver microsomes (< 0.001 nmol/min/mg). We have determined that both rat and human livers metabolize both CMEPA to MEA (0.308 nmol/min/mg and 0.541 nmol/min/mg, respectively) and CDEPA to DEA (0.350 nmol/min/mg and 0.841 nmol/min/mg, respectively). We have shown that both rat and human liver microsomes metabolize MEA (0.035 nmol/min/mg and 0.069 nmol/min/mg, respectively) and DEA (0.041 nmol/min/mg and 0.040 nmol/min/mg, respectively). We have also shown that the cytochrome P450 isoforms responsible for human metabolism of acetochlor, butachlor, and metolachlor are CYP3A4 and CYP2B6.

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: Characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat☆

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.

The production and modification of cytochrome P-450 difference spectra by in vivo administration of methylenedioxyphenyl compounds.

The in vivo administration of piperonyl butoxide (PB) or propyl isome (PI), both methylenedioxyphenyl compounds, to mice affects the levels of several hepatic cytochrome P-450 spectral interactions. The difference spectrum produced by carbon monoxide (commonly employed as a measurement of cytochrome P-450 levels) is initially lowered in magnitude by both compounds. The reduction is followed by an increase with time indicative of cytochrome induction. This biphasic effect, when produced by piperonyl butoxide, is accompanied by specific changes in the levels of the cytochrome P-450 difference spectra produced by hexobarbital, a type-I substrate, and pyridine, a type-II substrate. In addition, the ethyl isocyanide (EtNC) equilibrium point, calculated from the pH effect on the 455-nm and 430-nm peaks of the ethyl isocyanide-cytochrome P-450 difference spectrum, undergoes a biphasic shift. In contrast, propyl isome has the same effect on the substrate difference spectra as it does on the cytochrome level and produces no change in the ethyl isocyanide equilibrium point with time. Piperonyl butoxide, administered in vivo, produces a cytochrome P-450 difference spectrum when dithionite-reduced microsomes from treated animals are compared at different pH values. This spectrum, when observed by comparing microsomes from treated and untreated animals, has two pH-dependent Soret peaks and is essentially identical to the NADPH-dependent piperonyl butoxide-cytochrome P-450 spectrum produced by in vitro treatment. This spectrum is also produced in vitro by propyl isome and sulfoxide.

Induction of hepatic mixed function oxidases by the insecticide, mirex

Abstract the effect of mirex (dodecachlorooctahydro-1,3,4-metheno-2 H -cyclobuta-[c,d]pentalene) on hepatic cytochrome P-450 and O -demethylase activity has been tested in rats and mice by both injection and feeding studies. Intraperitoneal injection of mirex at a dose rate of 10 mg/kg or higher, increased the hepatic microsomal cytochrome P-450 level in mice. Under the same conditions 25 mg/kg were necessary to elicit the same response in the rat. The O -demethylase activity was also increased. Dietary mirex caused induction of cytochrome P-450 in the rat at all levels tested (1–250 ppm for 14 days). Examination of type I and type II substrate difference spectra indicate that induction at higher levels (100–250 ppm for 14 days) was not only greater but that the cytochrome induced was qualitatively different from that induced at lower levels. Ultrastructural examination of the liver tissue after mirex feeding revealed a proliferation of the smooth endoplasmic reticulum.

Induction of hepatic mixed-function oxidase enzymes in adult and neonatal mice by kepone and mirex.

Abstract The effects of 1, 10, and 50 ppm dietary kepone on hepatic mixed-function oxidases of adult male mice were measured. After 14 days dose-dependent increases in liver weight, hepatic microsomal protein, cytochrome P-450, Type I and Type II ligand binding to cytochrome P-450 and O - and N -demethylation of p -nitroanisole and aminopyrine were observed. Induction was approximately two-fold for all parameters measured at the 50-ppm level. The results obtained suggest that kepone is a nonspecific inducer, the no-effect level for induction based on O -demethylase activity in mice being less than 10 ppm. Body weight gain decreased in mice treated with 50 ppm kepone for 14 days. The effects of kepone and mirex fed to the mother after parturition on hepatic tissue of male and female neonatal mice were also measured. It is suggested that the increases observed in O - and N -demethylation of p -nitroanisole and aminopyrine in hepatic tissue from neonates result from the transfer of kepone and mirex through the milk. Induction in neonates was greater than that observed in adult male mice treated with kepone.

Oxidation of thiobenzamide by the fad-containing and cytochrome P-450-dependent monooxygenases of liver and lung microsomes

Two distinct microsomal pathways involved in the metabolism of thiobenzamide to thiobenzamide S-oxide have been identified and quantitated in the liver and lungs of mice and rats, using a highly inhibitory antibody against NADPH-cytochrome P-450 reductase. Approximately 50 and 65% of the oxidation in mouse and rat liver microsomes, respectively, was due to the FAD-containing monooxygenase, the remainder being catalyzed by cytochrome P-450. In the mouse lung, S-oxidation was predominantly via the FAD-containing monooxygenase while that in the rat lung was about 60% via the FAD-containing enzyme and 40% via cytochrome P-450. Cytochrome P-450-dependent S-oxidation of thiobenzamide was induced in the liver by treatment of mice with phenobarbital and slightly increased by treatment with 3-methylcholanthrene, while in rat liver either of these treatments caused only a small increase in metabolism due to cytochrome P-450. Thermal inactivation of the FAD-containing monooxygenase left the cytochrome P-450 component essentially unchanged. Thermally treated microsomes had a pH activity profile characteristic of cytochrome P-450 and were less inhibited by methimazole and thiourea when compared to untreated microsomes. Female mouse liver microsomes had a much higher, and female rat liver microsomes a lower, ability to S-oxidize thiobenzamide when compared to the males.