Steven A. Atlas

Species differences in the substrate specificity of hepatic cytochrome P-448 From polycyclic hydrocarbon-treated animals☆

Abstract The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N -hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N -hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N -hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N -hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.

Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin☆

Abstract Aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity is induced in cultured human lymphocytes by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) at a concentration in the growth medium 40 to 60 times less than the concentration of 3-methylcholanthrene (MC) necessary for maximal hydroxylase induction. In cultured lymphocytes from 19 individuals, the extent of hydroxylase induction by TCDD or MC ranged between 1.7- and 2.9-fold. Those individuals having (presumably under genetic control) lower basal and MC-inducible hydroxylase activities in their lymphocytes also have lower TCDD-inducible hydroxylase activity. Because of the day-to-day experimental variability, the variations within each assay, and for several other reasons discussed, we suggest that the observed variance of expression of hydroxylase induction more closely fits a unimodal, polygenic ( i.e. 2 or more genes) pattern rather than the trimodal (single gene) form of inheritance proposed recently by Kellermann and coworkers.

Genetic association of increases in naphthalene, acetanilide, and biphenyl hydroxylations with inducible aryl hydrocarbon hydroxylase in mice.

Abstract The induction of four hepatic monooxygenase activities, naphthalene trans-1,2-dihy-drodiol formation, acetanilide 4-hydroxylase, biphenyl 4-hydroxylase, and biphenyl 2-hydroxylase, by polycyclic aromatic compounds is genetically associated with the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity and cytochrome P 1 -450 in C57BL/6N and DBA/2N inbred mice and among progeny from the appropriate genetic crosses involving these two progenitor strains. These enzyme activities were studied with respect to (i) preferential inhibition by metyrapone, α-naphthoflavone, or Tween 80 in vitro ; (ii) use of the microsomal inducers 3-methylcholanthrene, β-naphthoflavone, 2,3,7,8-tetrachlorodibenzo- p -dioxin, or phenobarbital; (iii) apparent K m values; and (iv) heat inactivation. Several lines of evidence suggest that aromatic hydrocarbon-induced naphthalene monooxygenase, acetanilide 4-hydroxylase, biphenyl 4-hydroxylase, and biphenyl 2-hydroxylase activities are, like the induced aryl hydrocarbon hydroxylase activity, associated with cytochrome(s) P 1 -450 and that the basal activities of the first three of these enzymes are, like basal aryl hydrocarbon hydroxylase activity, associated with one (or more) forms of cytochrome P -450 other than cytochrome(s) P 1 -450. On the other hand, the basal biphenyl 2-hydroxylase activity in mouse liver appears to be associated solely with cytochrome(s) P 1 -450; this finding differs from all other basal monooxygenase activities associated with the Ah locus and studied thus far in a similar manner.

Genetically regulated aryl hydrocarbon hydroxylase induction in the eye: Possible significance of the drug-metabolizing enzyme system for the retinal pigmented epithelium-choroid

Abstract Aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction in the eye from inbred C57BL/6N (responsive) and DBA/2N (nonresponsive) mice and from offspring of the appropriate genetic crosses was investigated. The hydroxylase activity is two- to more than 10-fold inducible in the eye by intraperitoneally administered polycyclic hydrocarbons such as β-naphthoflavone, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin and is apparently under the same genetic regulation as that in liver. Histofluorescent examination of the eye from β-naphthoflavone-treated C57BL/6N mice indicates that the hydroxylase activity is closely associated with the retinal pigmented epithelium-choroid layer; the retina demonstrates little activity. It is proposed that genetically controlled induction of the hydroxylase activity and perhaps other cytochrome P-450-mediated mono-oxygenase activities in the retinal pigmented epithelium-choroid play an important role for detoxification and/or metabolic potentiation of drugs and other xenobiotics. These foreign compounds, which include drugs, insecticides, and chemical carcinogens, presumably enter the retina through the choriocapillaris.

The Ah locus: aromatic hydrocarbon responsiveness... of mice and men.

The membrane-bound cytochrome P-450-mediated monooxygenases are known to metabolize numerous drugs, environmental pollutants, chemical carcinogens, other foreign compounds, and numerous endogenous substrates (Thorgeirsson and Nebert, 1977; Ullrich et al. , 1977). The monooxygenase system-depicted in the upper left of Figure 1 as the rectangle of flavoprotein reductases, phospholipid, and hemoproteins-is deeply embedded in the endoplasmic reticulum membrane and perhaps in other membranes of the cell. The various monooxygenase ‘activities’ rely on the integrity of electron transport between the flavoproteins and the hemoproteins.