Biomedical subjects
Publications and source records attributed to E Hodgson.
In summary, it might be said that there is no simple answer to the problems inherent in the search for safer insecticides by rational methods. However, the subject has not yet been fully explored and opportunities remain for exploitation. The extent to which this is done will depend on more effective use of time, scientific manpower, and imagination.
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.
Induction of hepatic microsomal cytochrome P-450 in Dub:ICR male mice treated with phenobarbital, 3-methylcholanthrene, safrole, isosafrole, 5-tert.-butyl-1,3-benzodioxole (BBD), 2-methyl-5-tert.-butyl-1,3-benzodioxole (MBBD), and 2,2-dimethyl-5-tert.-butyl-1,3-benzodiozole (DBBD) was evaluated by measuring the cytochrome P-450 content, Type II:Type 1 binding ratio, ethylisocyanide pH equilibrium point, biphenyl 2- and 4-hydroxylase, ethylmorphine N-demethylase, ethoxyresorufin O-deethylase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Safrole and isosafrole treatment of mice produced a phenobarbital-type induction. BBD, but not MBBD and DBBD, induced cytochrome P-450 and formed a Type III metabolite-cytochrome P-450 complex, in vitro and in vivo. SDS-PAGE revealed that DBBD does induce proteins other than cytochrome P-450. These data suggest that the methylene carbon plays an important role in cytochrome P-450 induction.
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Hexose-6-phosphate dehydrogenase (H6PD) activity in washed hepatic microsomes from male ICR mice, when assayed with NADP+ and deoxyglucose-6-phosphate, was partially latent. Brief sonication or detergents activated H6PD causing an approximately 4- and 8.5-fold increase in NADPH generation respectively. The sonicated microsomes exhibited H6PD-linked N-demethylase activity toward aminopyrine. This activity was best sustained in the presence of deoxyglucose-6-phosphate, while galactose-6-phosphate, glucose-6-phosphate, and glucose were less effective. Reaction media containing sonicated microsomes, NADP+ and deoxyglucose-6-phosphate also catalyzed N-demethylation of p-chloro-N-methylaniline, N,N-dimethylaniline and nicotine, O-demethylation of p-nitroanisole, p-hydroxylation of aniline, ring hydroxylation of biphenyl at the 2- and 4-positions, dearylation of parathion, and the N-oxidation of N,N-dimethylaniline. In general, the hexose-6-phosphate dehydrogenase-linked monooxygenation rates were 60% or more of those observed in the presence of exogenous NADPH.
In the presence of NADPH and under aerobic conditions, thioether-containing organo-phosphorus and carbamate pesticides were oxidized by the FAD-dependent monooxygenase (EC 1.14.13.8) purified from pig liver microsomes. The stoichiometric relationship between NADPH and substrate during the course of the reaction was 1:1, and the product, in the case of disulfoton and phorate, was the sulfoxide. The product was optically active and further oxidation to the sulfone was not apparent. Furthermore, the sulfoxides of disulfoton, phorate and croneton were not substrates for this enzyme. n-Octylamine, a known cytochrome P-450 inhibitor, increased the rate of sulfoxidation reactions catalyzed by the FAD-dependent monooxygenase. Structure-activity relationships were investigated using thirty-nine possible substrates. Structural changes around the thioether sulfur that affect nucleophilicity or that cause steric hindrance tended to decrease the sulfoxidation rats. With phosphorodithioates, changes around the phosphorus atom also affected oxidation of the thioether sulfur. Although neither the thiono nor the thiol sulfur atoms were attacked, substitution of either sulfur by oxygen decreased sulfoxidation. Thioether-containing O, O-dimethyl phosphorodithioates were not oxidized as readily as their O, O-diethyl analogs. Tetram and its analogs, which contain a teritiary amine group, were also substrates for this enzyme, presumably forming the N-oxide.
1. In a preceding paper evidence was presented for the endogenous reduction of NAD(P)+ by mammalian hepatic microsomes and the concomitant reduction of cytochrome b5. The experiments reported here demonstrate that low concentrations of n-octylamine, in the presence of limiting quantities of NAD+, cause an increased level of cytochrome b5 reduction by mouse hepatic microsomes and also delays its reoxidation. 2. These effects are both NAD+ and n-octylamine dependent and appear to be due to an activation of the microsomal enzyme causing endogenous reduction of NAD(P)+ and also, in part, to inhibition of the autooxidation of reduced cytochrome b5. 3. Protection from the inhibitory action of sulfhydryl reagents on NADH-cytochrome b5 reductase was also observed in the presence of n-octylamine. 4. The results suggest that the enzyme(s) involved in the endogenous reduction of NAD(P)+ is not the microsomal alcohol dehydrogenase.
1. An enzyme system is present in mouse hepatic microsomes which is capable of reducing NAD+ and NADP+ and, consequently, of reducing cytochrome b5 in the absence of other externally added cofactors. 2. A similar enzyme system is also present in hepatic microsomes of rat and rabbit. 3. The rates of reduction of cytochrome b5 and auto-oxidation of reduced cytochrome b5 were, however, much slower in NAD+ supplemented reaction media than in those supplemented with NADH. 4. The effects of sulfhydryl reagents, chelating agents and divalent cations are reported.
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The cytochrome P-450-dependent monooxygenase system catalyzes a wide variety of oxidations of pesticide chemicals and related compounds. These reactions include epoxidation and aromatic hydroxylation, aliphatic hydroxylation, O-, N- and S-dealkylation, N-oxidation, oxidative deamination, S-oxidation, P-oxidation, desulfuration and ester cleavage and may result in either detoxication or activation of the pesticide. The current status of such reactions, relative to the production, in vivo, of biologically active intermediates in pesticide metabolism is summarized. More recently we have shown that the FAD-containing monooxygenase of mammalian liver (E.C.1.14.13.8), a xenobiotic metabolizing enzyme of broad specificity formerly known as an amine oxidase, is involved in a variety of pesticide oxidations. These include sulfoxidation of organophosphorus insecticides such as phorate and disulfoton, oxidative desulfuration of phosphonate insecticides such as fonofos and oxidation at the phosphorus atom in such compounds as the cotton defoliant, folex. The relative importance of the FAD-containing monooxygenase vis-a-vis the cytochrome P-450-dependent monooxygenase system is discussed, based on in vitro studies on purified enzymes.
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Livers of mice previously treated with potential inducers were tested for effects on the microsomal cytochrome P-450-dependent monooxygenase system (including its constituent electron transport enzymes) and other enzymes. Microsomes were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pesticides, including chlorinated hydrocarbons, organophosphates, a carbamate, and an insecticide synergistic were tested for induction along with the well known inducers, phenobarbital and 3-methylcholanthrene. The results indicate that the pesticide inducers tested are of the phenobarbital type.
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The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of 'apparent absolute absorption spectra' indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains. Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains. Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.
The flavin adenine dinucleotide--dependent monooxygenase in mammalian hepatic microsomes plays a major role in the oxidative metabolism of thioether-containing pesticides. Thirty-four compounds were tested, and it was determined that organophosphorus insecticides such as disulfoton and phorate are rapidly oxidized by the purified enzyme to their corresponding sulfoxides. The enzyme does not catalyze the oxidation of the thiophosphoryl and thiol sulfur atoms of these or other phosphorothioates and phosphorodithioates, or the oxidation of the sulfoxide to the sulfone. Carbamates aldicarb and Croneton are also oxidized, but at a lower rate.
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