Karl J. Netter

The induction of hepatic microsomal metabolism in rats following acute administration of a mixture of polybrominated biphenyls

Abstract Firemaster BP6, a mixture of polybrominated biphenyls (PBBs), was administered to female Sprague-Dawley rats (170–180 g) as a single ip injection at 25 and 150 mg/kg. Other animals received phenobarbital (PB), 3-methylcholanthrene (3MC), or PB and 3MC together. Animals were killed at intervals of 12, 24, 48, 192, and 336 hr after treatment with PBBs, or 24 hr after PB, MC, or PB-MC, and various hepatic microsomal parameters were measured. After 150 mg/kg of PBBs, cytochrome P450 concentrations reached a maximum by 48 hr (225% of control), then remained elevated through 336 hr. A similar pattern of induction was observed for epoxide hydratase and aniline hydroxylase activities. In contrast, ethylmorphine- N -demethylase and ethoxycoumarin- O -deethylase activities initially increased to 256 and 1379% of control, respectively, at 48 hr, then continued to increase to 583 and 3197% of control at 336 hr. Ethylmorphine- N -demethylase was induced by PB (579% of control) but not 3MC, and ethoxycoumarin- O -deethylase was more sensitive to 3MC (3852% of control) than PB (891% of control). Arylhydrocarbon hydroxylase activity was 1531% of control 192 hr after 150 mg/kg of PBBs which was similar to the activity induced by PB-3MC (1851% of control) but less than that induced by 3MC alone (3499% of control). No significant increases were observed in any parameters until 192 hr after treatment with 25 mg/kg of PBBs. The enzyme activities and the wavelength of maximum absorption for the cytochrome P450-CO complex from microsomes of animals treated with PBBs indicated that the PBBs produced a “mixed” type of hepatic microsomal enzyme induction with inducing properties of both PB and 3MC.

Studies on the interaction of safrole with rat hepatic microsomes

Abstract (1) Similar to previous results with methylenedioxyphenyl compounds microsomes from safrole pretreated rats showed, on reduction with NADH, NADPH or Na 2 S 2 O 4 , characteristic absorption maxima at 427 and 455 nm. The same spectrum can be obtained after incubation in vitro of control microsomes with safrole, NADPH and oxygen. (2) Subsequent addition of carbon monoxide to microsomes of safrole pretreated rats causes an absorption maximum at 448 nm, characteristic of the 3-methylcholanthrene type of induction of microsomal hydroxylase protein. (3) The suspected cytochrome P-450-safrole metabolite complex, which can be visualized only in the reduced state of cytochrome P-450, is very stable as witnessed by its preservation through the preparation procedure for microsomes or after dialysis or detergent treatment. However, when safrole or ethylbenzene is added, both absorption maxima decrease in a time dependent manner. This can be measured for each time point after complete reduction of the microsomal preparation by adding Na 2 S 2 O 4 . (4) From this it is concluded that the carcinogen safrole leads to the biosynthesis of a 3-methylcholanthrene type cytochrome P-450 and formation of a safrole metabolite-cytochrome P-450 complex which in turn can be cleaved in vitro by safrole or other lipophilic compounds.

A novel haemoprotein induced by isosafrole pretreatment in the rat.

Abstract Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to demonstrate that pretreatment of rats with isosafrole results in the formation of a novel species of cytochrome P-450 (mol. wt. 54,000) quite distinct from that induced by phenobarbitone pretreatment (mol. wt. 50,000) or 3-methylcholanthrene (mol. wt. 58,000).

7-alkoxyquinolines: New fluorescent substrates for cytochrome p450 monooxygenases

A series of 7-alkoxyquinolines was synthesized and tested as substrates with hepatic microsomes prepared from male Wistar rats. Microsomal O-dealkylation rates and kinetic constants were determined for the 7-alkoxyquinolines with microsomes from control, 3-methylcholanthrene (MC)-pretreated, and phenobarbitone (PB)-pretreated rats. Structure-activity relationship studies indicated that the 7-benzyloxyquinoline was the most rapidly metabolized substrate for control microsomes and those from PB-pretreated rats, whereas the 7-ethoxy- and 7-propoxyquinolines were O-dealkylated more rapidly by microsomes of MC-pretreated animals. Differences in activities occurred in Vmax and apparent Km values; however, there does not appear to be a correlation between these two values for the different quinoline substrates. Apparent Km and Vmax values for the 7-alkoxyquinolines were: control microsomes, Km = 71-773 microM, Vmax = 0.37-8.4 nmol 7-quinolinol/min/mg protein; MC microsomes, Km = 0.5-14 microM, Vmax = 0.29-2.7 nmol 7-quinolinol/min/mg protein; PB microsomes, Km = 2.8-46 microM, Vmax = 0.9-12 nmol 7-quinolinol/min/mg protein. All of the quinoline substrates gave Type I binding spectra with control and MC microsomes. With PB microsomes, Type I. Reverse Type I, and a mixture of the two types of binding spectra were observed. Comparisons of the structure-activity relationships, levels of induction, and kinetic constants were made with 7-alkoxycoumarin and 7-alkoxyphenoxazone analogs. In addition, three new coumarin substrates (7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin) are described.

Ethoxyquin as an inducer and inhibitor of phenobarbital-type cytochrome P-450 in rat liver microsomes.

Abstract The effect of ethoxyquin in vivo and in vitro on drug metabolism in rat liver microsomes was studied. In feeding experiments, a threshold dose of induction was found at 0.05% ethoxyquin for 14 days. At 0.5% ethoxyquin, relative liver weight, cytochrome P -450 content, cytochrome b 5 content, ethylmorphine demethylation, and ethoxycoumarin deethylation were increased by a factor of 1.5 to 2. Aryl hydrocarbon hydroxylase activity was, however, not induced but even decreased by 0.5% ethoxyquin in food. Induction of epoxide hydratase was marked, amounting to 400% of control after 0.5% ethoxyquin. The induced enzyme was similar to the phenobarbital-inducible cytochrome P -450 in its CO spectrum, in its affinity for the ligand metyrapone, in the preferential inhibition of monooxygenase activity by metyrapone and not α-naphthoflavone, and in the increase in the phenobarbital-inducible band after gel electrophoresis of microsomal proteins. Mixed pretreatment with ethoxyquin and 3-methylcholanthrene led to formation of cytochrome P -448. However, monooxygenase activities were slightly lower and epoxide hydratase activity was considerably higher than after treatment with 3-methylcholanthrene alone. Ethoxyquin inhibited benzo( a )pyrene hydroxylation and ethoxycoumarin deethylation in vitro . These reactions were less sensitive to ethoxyquin in 3-methylcholanthrene-stimulated microsomes than in phenobarbital-stimulated microsomes, indicating that ethoxyquin does not only preferentially induce but also preferentially inhibits phenobarbital-type cytochrome P -450.

Heterogeneity of carbonyl reduction in subcellular fractions and different organs in rodents

The pattern and distribution of carbonyl reduction in liver, kidney and adrenal gland subcellular fractions of NMRI mice, Wistar rats and Hartley guinea pigs were examined using the ketone compound metyrapone (2-methyl-1,2-di(3-pyridyl)1-propanone) commonly used as a diagnostic cytochrome P450 inhibitor. A direct HPLC method for alcohol metabolite determination instead of the indirect spectrophotometric recording of pyridine nucleotide oxidation at 340 nm was applied. All the tissues examined in these species rapidly reduced the employed compound but at the subcellular level no general distribution scheme of specific activity was found, although in all fractions metyrapol formation could be attributed to aldo-keto reductases. Cytosolic and microsomal metyrapone reducing enzymes are distinguished by their inhibitor sensitivity to phenobarbitone and quercitrin and thus can be characterized as aldehyde and ketone reductases according to the inhibitor subclassification of the aldo-keto reductase family. Moreover, the enzymes also differ with respect to their immunological cross-reactivity to anti-microsomal mouse liver metyrapone reductase antibodies. Immunological homologies were found between metyrapone reductases of liver microsomes from all species and kidney and adrenal gland microsomes from guinea pig. However, the protein of all the cytosolic fractions as well as that of kidney and adrenal gland microsomes from mouse and rat did not cross-react with the antibodies, indicating the absence of common antigenic determinants. From catalytic properties and functional data it is concluded that hydroxysteroid dehydrogenases present in the suspected subcellular fractions form a structurally and functionally related enzyme family which may have been conserved during evolution.

Atypical inductive properties of rifampicin

Abstract Hepatic and microsomal parameters, metabolism of cytochrome P-450-dependent substrates and spectral properties of cytochrome P-450 have been used in mice in order to classify rifampicin as an inducer by comparing it to phenobarbitone and 3-methylcholanthrene. Rifampicin significantly enhanced relative liver weight, cytochrome P-450 content, microsomal protein, weight of the 100,000 g pellet and shortened the zoxazolamine paralysis time. Compared on the basis of microsomal protein, of five substrate reactions only the ethylmorphine demethylation was enhanced; ethoxycoumarin deethylation and biphenyl 1–2-hydroxylation were unaltered; ethoxyresorufin de-ethylation and biphenyl-4-hydroxylation were decreased. The CO-cytochrome P-450 absorption maximum showed a blue shift of about 0.5 nm after only 1 day of rifampicin pretreatment, while there was no significant blue shift after 1 day of 3-methylcholanthrene pretreatment. Rifampicin has been shown to be an inducer in NMRI mice. Although resembling phenobarbitone it seems, however, to be a similarly atypical inducer as it is in man.

Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts.

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.

On the inhibition of microsomal drug metabolism by SKF 525-A☆

Abstract Kinetic experiments have been carried out on the inhibition of the O-demethylation of p-nitroanisole and the N-demethylation of N-monomethyl-p-nitroaniline by β-diethylaminoethyl-diphenyl-n-propylacetate-HCl (SKF 525-A). The source of the enzyme were liver microsomes of male mice pretreated with phenobarbital. Addition of original SKF 525-A to the reaction mixture resulted in an apparently non-competitive inhibition of both demethylation reactions. When the substance was recrystallized from benzene the non-competitive type of inhibition was converted to an apparent competitive type of inhibition. Use of other solvents for recrystallization such as water, methanol, cyclohexane and chlorobenzene did not lead to a change of the type of inhibition. Therefore recrystallization from benzene seemed to be unique in causing this peculiar change of kinetic behaviour. Benzene produces no chemical alteration of the SKF 525-A molecule that could be detected by a number of physicochemical methods employed.

Stimulation of hepatic microsomal metabolism in mice by a mixture of polybrominated biphenyls

The pattern of stimulation of hepatic microsomal mixed function oxidases has been studied in female NMRI mice following a single ip injection of 150 mg/kg polybrominated biphenyls (PBBs) and compared to the patterns produced by phenobarbital (PB), 3 X 100 mg/kg; 3-methylcholanthrene (MC), 3 X 20 mg/kg; and these two agents administered together at these doses. Cytochrome P450, ethylmorphine N-demethylase, and epoxide hydratase reached maximums of 200, 200, and 350% of control values, respectively, at 48 hr after treatment with PBBs. Ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase were maximally increased to 400 and 180% of control values, respectively, 96 hr after PBBs. The reduced cytochrome P450 ethylisocyanide difference spectra and the inhibition of ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase activity by metyrapone and alpha-naphthoflavone indicated that the characteristics of the cytochrome P450 and the cytochrome P450-dependent enzymes changed with time after administration of PBBs. These results indicate that the enzyme-stimulating properties of PBBs alter, changing from PB-like to MC-like, with time after administration. These findings provide an explanation for the effects of PBBs on the toxicity of bromobenzene, indicating that PBBs represent a new and previously unrecognized calss of toxicity-modifying agents sharing properties of both PB and MC.