Liver Aryl Hydrocarbon Hydroxylase Research Articles

Effects of indole and tryptophan on cytochrome P-450, dimethylnitrosamine demethylase, and arylhydrocarbon hydroxylase activities

The effects of indole and l-tryptophan feeding on the activities of two liver microsomal enzymes, dimethylnitrosamine demethylase (DMN-d) and arylhydrocarbon hydroxylase (AHH), were studied in rats and hamsters. Both indole and tryptophan increased cytochrome P-450 concentration and DMN-d activity in rats, but only indole was effective in hamsters. AHH activity in both species was induced by indole. Both indole and tryptophan increased the ratio A 392–500nm/ A 410–500nm obtained by n-octylamine difference spectroscopy of rat liver microsomes but not of hamster liver microsomes. Indole increased the absorbance difference ratio A 455–500nm/ A 430–500nm of rat liver cytochrome P-450 obtained by ethyl isocyanide. The opposite was true for hamsters, which showed a low band at 455 nm. Indole gave a type II binding spectrum with rat liver microsomes, but the values for absorption minimum and maximum for tryptophan were somewhat outside the reported values for type II binders. Indole which induced liver AHH activity, protected against 7,12-dimethylbenz[a]anthracene (DMBA)-produced mammary gland carcinogenicity, whereas tryptophan was without effect.

Altered activities of hepatic and extrahepatic microsomal mixed function oxidase enzymes in diabetic and adrenalectomized diabetic rats.

Microsomal aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECD) activities were determined in liver, lung and intestine of control, streptozotocin(STZ)-diabetic, and diabetic-adrenalectomized male and female rats. Hepatic cytochrome P-450 content was also determined. The diabetic state reduced hepatic AHH activity and increased ECD activity in control male rats, and failed to do so in the adrenalectomized male rats. The diabetic state increased hepatic AHH and ECD activities in the control female rats, while in the adrenalectomized female rats the activities of both enzymes were decreased compared to the control diabetic rats. STZ-induced diabetes produced a decrease in pulmonary AHH and ECD activities and increased intestinal AHH and ECD activities in both sexes. The diabetic state in the adrenalectomized rats resulted in further reduction in pulmonary AHH and ECD activities in both sexes, but failed to increase the intestinal AHH activity only in the female rat. Hepatic cytochrome P-450 contents were increased in the female but not male adrenalectomized rats when treated with STZ. The effects of STZ-induced diabetes in adrenalectomized rats on AHH, ECD and hepatic cytochrome P-450 content in liver, lung and intestine depended upon sex of animal, substrate and tissue.

Benzo[ a]pyrene metabolism and macromolecular binding in strains of Ah responsive and Ah non-response mice

Using liver microsomes as the enzyme source in in vitro assays, benzo[ a]pyrene (BP) metabolism was studied in eight inbred strains of mice (C57BL/6J, DBA/2HaD, BALB/cCR, AKR/Sn, RF/J, CBA/J, C57L/J and 129/J). BP metabolite formation was monitored by high pressure liquid chromatography (HPLC) and by following aryl hydrocarbon hydroxylase (AHH) activity. Other parameters measured included the formation of alkali-extractable radioactivity due to [ 3H]BP metabolites, microsomal protein binding, DNA binding and epoxide hydrase activity. The induction of BP metabolism and binding to macromolecules was studied after treatment of animals with phenobarbital (PB) or 3-methylcholanthrene (MC). Four of the mouse strains (C57BL/6J, BALB/cCr, CBA/J and C57L/J) were highly inducible with respect to liver AHH when pretreated with MC. The induction of AHH by MC in these strains correlated well with the radioactive metabolites of [ 3H]BP remaining in the alkali extract derived from the AHH assay mixture and with the increased binding of [ 3H]BP to microsomal protein and DNA. In addition to the eight strains listed above, eight recombinant inbred lines also showed a positive correlation between AHH induction and induction of DNA-binding metabolites. PB pretreatment resulted in less than two-fold induction of AHH and alkali-extractable radioactivity. However, DNA and microsomal protein binding were induced by PB pretreatment more than AHH. Ratios of MC-induced/basal activity for BP-phenols were very similar to induction ratios of AHH activity determined by the fluorometric method. BP-quinone formation was induced to the same extent as phenols. This relationship did not hold for dihydrodiol formation; dihydrodiol induction was often higher than AHH or phenol induction. For MC-pretreated mice, dihydrodiol induction, as determined by HPLC, did not parallel macromolecular binding induction as closely as did AHH. For PB-pretreated mice, dihydrodiol induction was as poor an indicator of binding induction as AHH. Epoxide hydrase activity, using styrene oxide as substrate, was induced markedly by PB-pretreatment, but very little by MC-pretreatment. Epoxide hydrase induction did not parallel BP-dihydrodiol induction when microsome preparations from MC- or PB-treated mice were used. These data suggest this enzyme is not rate limiting in this system.

Aryl Hydrocarbon Hydroxylase and Epoxide Hydratase Activities: Age Effects in Mammary Epithelial Cells of Sprague-Dawley Rats<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

By use of techniques developed for rat mammary epithelial cell isolation, studies were conducted to characterize aryl hydrocarbon hydroxylase (AHH) and epoxide hydratase (EH) activities and to determine whether changes in the enzyme activities might contribute to the age-dependent changes in polycyclic aromatic hydrocarbon (PAH)-induced tumorigenesis in the rat mammary gland. Constitutive AHH and EH activities in rat mammary epithelial cell homogenates were substantially lower than those measured in liver (9,500×g) supernatant. The administration of 3-methylcholanthrene (MCA) or 7,12-dimethylbenz[a]anthracene increased mammary epithelial AHH activity 93- and 44-fold, respectively, whereas hepatic AHH levels rose 17- and 6-fold. Neither inducer had any effect on constitutive EH activity in either tissue. The addition (0.1 mM) of 7,8-benzoflavone (7,8-BF), SKF 525-A, or metyrapone to homogenates of mammary epithelial cells from MCA-treated rats inhibited AHH activity 93, 48, and 22%, respectively. In contrast, 7,8-BF addition decreased rat liver AHH activity 83%, whereas SKF 525-A and metyrapone had little effect. EH activities in rat liver and mammary epithelial cells were reduced with addition of 1,1,1-trichloro-2-propene oxide and cyclohexene oxide; however, trans-stilbene oxide had no effect in either tissue preparation. Additional studies determined the age-dependent changes in rat mammary epithelial AHH and EH activities. Constitutive AHH activity remained unchanged in rats between 40 and 100 days of age. In contrast, mammary epithelial EH activity was highest in 40-day-old Sprague-Dawley rats, declined in rats between 40 and 60 days of age, and remained constant in rats between 60 and 100 days of age. The level of MCA-induced AHH activity in rat mammary epithelial cell homogenates was age-dependent. MCA administration induced constitutive AHH activity 85- and 74-fold in mammary epithelial cells isolated from 50- and 60-day-old Sprague-Dawley females. The magnitude of mammary epithelial AHH induction was significantly lower in both younger and older animals. The results demonstrated PAH-metabolizing enzymes in rat mammary epithelial cells and suggested that the age-dependent incidence of mammary tumorigenesis may be attributed in part to the age-dependent inducibility of AHH activity.

Effects of polyhalogenated aromatic hydrocarbons on benzo(a)pyrene hydroxylase activity in the intestine and liver

Aryl hydrocarbon hydroxylase (AHH) has been measured as benzo(a)pyrene hydroxylase in the intestine and liver of rats and mice treated with a single dose of different polyhalogenated aromatic hydrocarbons. Maximal stimulation of liver AHH activity is reached with a dose of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) half as great as that necessary for maximal stimulation of the intestine. The duration of the effect of TCDD on intestinal AHH differs from the constant increase that occurs in the liver. Although the magnitude of the stimulation by 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) is less than that of TCDD, the qualitative changes in intestinal and liver AHH are similar. The changes in activity of intestinal and hepatic AHH were not directly correlated in the tissues of rats treated with several other polyhalogenated aromatic hydrocarbons. Liver and intestinal AHH activity were affected differently by fasting. These results suggest that AHH activity in the intestine and liver has different control mechanisms.

Mutagenicity and metabolism of dimethylnitrosamine and benzo[α]pyrene in tissue homogenates from inbred syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[α]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SsLak, LSH/SsLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. Dimethyl-nitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[α]pyrene as substrate. MC does not induce AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC.

Differential inhibition of aryl hydrocarbon hydroxylase in human foetal liver, adrenal gland and placenta*.

Abstract It is known from experiments on rats and mice that control and polycyclic aromatic hydrocarbon‐induced activities of aryl hydrocarbon hydroxylase differ profoundly with regard to their inhibition by several compounds. In order to obtain information about the nature of the human enzyme system from different tissues, the inhibition of aryl hydrocarbon hydroxylase (AHH) by different compounds in the human foetal and adult liver, foetal adrenal gland, as well as the placenta was studied. Substantial AHH activity was present in all the livers and adrenal glands, but only in term placentas from smoking mothers. The placental AHH was inhibited by 7,8‐benzoflavone, but not appreciably by SKF 525 A, aminopyrine or metyrapone. On the other hand, the foetal liver AHH was inhibited by SKF 525A, aminopyrine and metyrapone, whereas 7,8‐benzoflavone apparently activated the enzyme. The adult human liver AHH resembled that of foetal liver. The foetal adrenal AHH displayed a pattern of inhibition differing from both the liver and the placenta. Maternal cigarette smoking did not have a substantial effect either on the enzyme activity or on the inhibitory properties. Foetal hepatic AHH resembled the enzyme from control‐rat liver and the placental AHH was similar to the enzyme from 3‐methylcholanthrene‐treated rat liver. The effect of 7,8‐benzoflavone on control rat liver AHH activity varied greatly from the prenatal to the adult period, whereas AHH activity in MC‐treated rat liver was always strongly inhibited by 7,8‐benzoflavone.

Multiple forms of cytochrome P-450: Resolution and purification of rabbit liver aryl hydrocarbon hydroxylase

We describe the resolution and partial purification of two minor forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo- p -dioxin. Both forms have different electrophoretic mobilities when compared to the major form of cytochrome P-450 isolated from this source. The two cytochromes show different activities with several substrates. One form is very active in the hydroxylation of benzo( a )pyrene when reconstituted with highly purified NADPH-cytochrome P-450 reductase.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

Effect of benzo(a)pyrene and chlorpromazine on aryl hydrocarbon hydroxylase activity from rat tissues

The effects of 3,4-benzo(a)pyrene (BP) and chlorpromazine (CP) on th e distribution and relative levels of aryl hydrocarbon hydroxylase (AHH) in rat tissue were studied in vitro. The metabolism of AHH in BP-treated lymph node, lung, prostate, kidney, salivary glands, liver, spleen and mammary gland was markedly increased, while only lung, liver, and kidney tissue showed a marked increase in CP experiments. Prior to treatment, the metabolism of polycyclic hydrocarbons occurred primarily in the liver. However, kidney and lung tissues became much more important in metabolizing these hydrocarbons after exposure to BP or CP. The importance of AHH activity in protective tissues of entry into the body is discussed.

Simple vs. complex inheritance of inducible aryl hydrocarbon hydroxylase in mouse tissues.

The genetics of induction of hepatic and lung aryl hydrocarbon hydroxylase (AHH) have been studied in Af/Ki and AKR/Ki mice and in their F1 and F2 progeny after administration of 3-methylcholanthrene (3MC). Furthermore, the induction of AHH was investigated using the fetal liver explant model system with 3MC, trans-1,2-dihydroxy-3MC, and 4'-bromoflavone as the inducers. The results obtained with the above strains were contrasted with those from the C57BL/6Ki, DBA/2+Ki, and their crosses. The present investigation revealed a complex pattern of inheritance of basal and inducible AHH in lung and liver of AKR/Ki and Af/Ki, with a poor correlation between lung and liver. Hepatic AHH was not fully inducible in the F1 hybrids, while the frequency distribution function in the F2 mice was suggestive of more than two distinct classes.

Selection of inducers: An important factor in characterizing genetic differences to induction of aryl hydrocarbon hydroxylase in strains of mice

The effect of various microsomal enzyme inducers such as DDT, benzpyrene, 3-MC, TCDD or phenobarbital on liver microsomal mixed-function oxidases and cytochrome P450 content in mice genetically responsive (C57B1/6J) and resistant (DBA/2J) to induction of aryl hydrocarbon hydroxylase (AHH) was studied. 3-MC and benzpyrene administration stimulated liver AHH activity 6–8 fold in C57B1/6J mice but had no effect in DBA/2J mice. However, intraperitoneal administration of TCDD increased AHH activity in both C57BL/6J and DBA/2J mice. This increase was accompanied by shift in the peak of cytochrome P450 difference spectrum from 450 to 448 nm. It is concluded that genetic resistance to AHH stimulation in DBA/2J mice is influenced by the type of inducer used.