Microsomal Mixed-function Oxidase Activity Research Articles

Control of the microsomal mixed-function oxidase by Ox 2 and Ox 5 genes in houseflies

The microsomal mixed-function oxidase (MFO) in houseflies is controlled by two semidominant genes, Ox 2 and Ox 5 , situated on chromosomes 2 and 5, respectively. MFO controlled by these genes has almost similar affinity toward cyclodiene epoxidation, but only the one controlled by Ox 5 can degrade DDT. A strain, YFc, homozygous for both oxidase genes shows twice as much MFO activity toward aldrin as either of the parent homozygotes, Fc or Y, but only as much activity toward DDT as the parent strain Fc. These Ox 2 and Ox5 genes in the YFc strain maintain their identity with regard to DDT in their hybrids with a susceptible homozygous strain recessive for the two oxidases as seen by segregation in the test-cross progenies. The Ox 2 gene is situated at 32 units from the Deh and car genes, 40 units from stw, and about 69 units from Mk. The Ox 5 gene is situated at 40 units from the ocra gene and 82 units from apt on chromosome 5.

Ultrastructure and Drug Metabolism in the Developing Guinea Pig

Unlike a variety of other animal species, such as the rabbit, mouse or rat, the guinea pig has a relatively long gestation period and is a more fully developed animal at birth. Kuenzig et al. reported that drug metabolic activity which increases very slowly during fetal life, increases rapidly after birth. Hepatocytes of a 3-day old neonate metabolize drugs and reduce cytochrome P-450 at a rate comparable to that observed in the adult animal. Moreover the administration of drugs like phenobarbital to pregnant guinea pigs increases the microsomal mixed function oxidase activity already in the fetus.Drug metabolic activity is, generally, localized within the smooth endoplasmic reticulum (SER) of the hepatocyte.

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b 5, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase ( V max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.

Exogenous factors affecting polycyclic hydrocarbon hydroxylase activity

In a number of experiments it has been shown that induction of increased microsomal mixed-function oxidase activity results in inhibition of carcinogenesis due to exposures to a variety of chemical carcinogens. Thus, this enzyme system has the capacity to exert a protective effect against chemical carcinogens. Substantial studies of the distribution of one component of the microsomal mixed function oxidase system, the polycyclic hydrocarbon hydroxylases, have been carried out. In addition to other tissues, polycyclic hydrocarbon hydroxylases occur in the major portals of entry. Their activity in the lung and small intestine is quite labile, being altered by diet as well as defined chemical inducers. Present data indicate that most and possibly all of the polycyclic hydrocarbon hydroxylase activity in these two tissues is due to exposure to exogenous inducers. Studies of 3-MMAB N-demethylase in the small bowel show a similar type of control. It is difficult at the present time to assess the role of the exogenous control of the mixed function oxidase system in preventing noxious effects from toxic chemicals in the environment. It could be of considerable importance to the health of man and other species.

Carbon tetrachloride-induced protection against carbon tetrachloride toxicity: The role of the liver microsomal drug-metabolizing system

Intragastric administration to rats of a small non-lethal dose of carbon tetrachloride produces a dramatic protective action against subsequently administered large and ordinarily lethal doses of the same liver poison. Resistance to the lethal effect of carbon tetrachloride sets in by 12 hr and is fully developed by 24 hr. Protection lasts for 3–5 days after which it gradually subsides. Activity of liver microsomal aminopyrine demethylase and cytochrome P-450 concentration were followed throughout the course of development of the protection phenomenon. Activity of aminopyrine demethylase and cytochrome P-450 concentration began to fall immediately after administration of the small initial dose of carbon tetrachloride. At 24 hr, when protection was complete, the activity of this enzyme system was about one-fourth of control levels. The depression lasted for 4 days, which was followed by gradual recovery. The exact parallelism between depression of liver microsomal mixed function oxidase activity and resistance to the lethal effects of carbon tetrachloride affords strong evidence for the view that metabolism of carbon tetrachloride by the liver microsomal drug-metabolizing system is a necessary prerequisite for the toxicity of this liver poison.

Mechanism of microsomal mixed-function oxidase inhibitor from the housefly Musca domestica L

An inhibitor of microsomal mixed-function oxidase activity from the head of the housefly, Musca domestica L., has been partially purified by chromatography on Sephadex G-25. Its chemical characteristics, in agreement with investigations from other laboratories, indicate that it is the eye pigment, xanthommatin. The inhibitory mechanism has been investigated using microsomal preparations from mouse liver and housefly abdomens as well as a purified microsomal NADPH-cytochrome c reductase from the housefly. Kinetic expriments on the rate of p-nitroanisole demethylation, NADPH oxidation, and cytochrome c reduction indicate that the inhibitor acts by accepting electrons at the reductase level thus impeding electron flow to cytochrome P-450. The inhibitor will accept electrons from the purified NADPH-cytochrome c reductase and also stimulate the reduction of cytochrome c by this enzyme. Spectral studies of cytochrome b 5 and cytochrome P-450 show that the inhibitor can prevent the reduction of cytochrome b 5, presumably by accepting electrons prior to the cytochrome level, but does not interact with cytochrome P-450 nor does it affect the binding of other substrates to this cytochrome.

Rate-limiting steps in drug metabolism

Comparative studies have been conducted of the activity of microsomal mixed-function oxidases from livers of normal, precirrhotic and cirrhotic rats linked with the metabolism of type-I (aminopyrine, hexobarbital), type-II (aniline, metyrapone) and “modified type-II” (corticosterone) substrates. The following factors were investigated : the possible role of cytochrome P-450 content, the state of the “substrate-binding protein” of this enzyme, the degree of affinity of this hemoprotein for both type-I and type-II substrates and finally, the activity of the enzymes of the microsomal electron-transport chain (both in the absence and in the presence of type-I substrate) — as rate-limiting reactions, “tight spots” in the biotransformation of drugs in experimental microsomes. It was found that the hydroxylation activity for type-II and “modified type-II” substrates during the entire period of liver cirrhosis development is determined by the cytochrome P-450 content and the amplitude of maximal spectral changes observed in the presence of excess substrate. Type-I substrate metabolism, however, is limited in the precirrhotic phase by the state of the“substrate-binding protein” contained in P-450 as well as by the NADPH-cytochrome P-450 reductase activity. On the other hand, the N-demethylating activity in CCl4-cirrhotic liver microsomes does not depend on either the concentration of P-450, on the amplitude of the maximal spectral changes or on the K8 value. The rate-limiting step in this case is the rate of reduction of the P-450-substrate complex by NADPH.

Response of hepatic microsomal mixed-function oxidases to various types of insecticide chemical synergists administered to mice

For several hours after a single intraperitoneal (i.p.) dose to mice, piperonyl butoxide, 2-methylpropyl 2-propynyl phenylphosphonate (NIA 16824), and 5,6-dichloro-l, 2,3-benzothiadiazole (WL 19255) inhibit hepatic microsomal mixed-function oxidase (mfo) activity and the apparent level of cytochrome P-450 but, after 24–72 hr, they induce increased mfo activity and P-450 content. Other types of insecticide chemical synergists studied are generally less active or inactive in this respect. The active compounds enhance the toxicity of several insecticide chemicals and markedly prolong hexobarbital sleeping time but not hexobarbital toxicity. The action of WL 19255 differs from that of piperonyl butoxide and NIA 16824 in that it is relatively non-specific in inhibiting enzymatic activity on several substrates, gives a more prolonged effect on P-450, and the magnitude of induction is greater.

On the use of nicotinamide in assays for microsomal mixed-function oxidase activity

Nicotinamide, an additive to almost all media used to study oxidative metabolism of substrates of hepatic microsomal mixed-function oxidase, is shown to inhibit the metabolism of known substrates of this system. Inhibition of aminopyrine demethylation appears to be competitive, while inhibition of aniline hydroxylation appears to be a mixture of competitive and noncompetitive inhibition. It is shown that an accurate assay of substrate metabolism can be obtained in the absence of nicotinamide if the assay time is kept short, NADPH concentration is maintained at a high level, and the concentration of microsomes in the medium is kept low.