AHH Activity Research Articles

Effects of antioxidants on metabolism of aromatic polycyclic hydrocarbons

A number of antioxidants have been shown to inhibit the neoplastic effects of a variety of chemical carcinogens. Some of these antioxidants such as BHA and BHT are widely distributed in the environment, and it is possible that they could play a role in diminishing the impact of exposures to chemical carcinogens. The mechanism or mechanisms of these inhibitions have not been established. Two major categories seem likely: (a) some type of direct interaction between antioxidant and reactive species of carcinogen and (b) an indirect effect. Using BHA as the antioxidant and BP as the carcinogen, studies of this latter possibility have been initiated. The metabolism of BP by liver microsomes in female A/HeJ mice fed a control diet or a diet containing BHA was studied. AHH activity was not changed by BHA feeding. However, the measurement of other parameters demonstrated that BHA feeding altered the microsomal system metabolizing BP. Incubation of BP and calf thymus DNA with liver microsomes from BHA-fed mice showed about half the binding of BP metabolites to DNA as compared to that of controls. The AHH activity of mice fed BHA was much more sensitive to in vitro inhibition by α-naphthoflavone than that of controls. The amount of cytochrome P-450 was increased per unit weight of microsomal protein and liver in mice fed BHA. The ethyl isocyanide binding spectra were measured to see if alterations of cytochrome P-450 might be produced by BHA feeding. The maximum at 430 mμ was the same in control and BHA-fed mice. However, the maximum at 455 mμ was lower in BHA-fed mice than in controls, which indicated that BHA had caused some change. These data show that BHA feeding results in altered properties of liver microsomes, including a decrease in BP metabolite binding to DNA.

Alterations in Microsomal Metabolism of Benzo [a] pyrene in Mice Fed Butylated Hydroxyanisole2

The metabolism of benzo[a]pyrene (BP) by liver microsomes in female A/HeJ mice fed a control diet or a diet containing butylated hydroxyanisole (BHA) was studied. Aryl hydrocarbon hydroxylase activity (AHH) was not changed by BHA feeding. However, the measurement of other parameters demonstrated that BHA feeding altered the microsomal system metabolizing BP. Incubation of BP and calf thymus DNA with liver microsomes from BHA-fed mice showed about half the binding of BP metabolites to DNA as compared to that of controls. The AHH activity of mice fed BHA was much more sensitive to in vitro inhibition by alpha-naphthoflavone than that of controls. The amount of cytochrome P450 was increased per unit weight of microsomal protein and liver in mice fed BHA. The ethyl isocyanide binding spectra were measured to see if alterations of cytochrome P450 might be produced by BHA feeding. The maximum at 430 nm was the same in control and BHA-fed mice. However, the maximum at 455 nm was lower in BHA-fed mice than in controls, which indicated that BHA had caused some changes. The data showed that BHA feeding resulted in altered properties of liver microsomes, including a decrease in BP metabolite binding to DNA.

Strain differences in aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene in rabbits.

We determined both basal and induced AHH activity in livers of six partially inbred strains of rabbits. Strain III rabbits had the highest enzyme activity upon induction by 3-MCA, i.e., four to five times that in strain WH (noninducible), which has the lowest enzyme activity. AHH induction was also "low" in strains X, OS, ACEP, and AC. F1 hybrids between strains III and WH revealed a differential response to the induction of liver AHH activity by MCA: the levels of induced hydroxylase activity were consistently higher in (III X WH)F1 rabbits than in the reciprocal (WH X III)F1 hybrids. All possible crosses between these two "extreme" strains are now being analyzed to estimate the number of genes involved in their response difference to MCA.

HEME SYNTHESIS AND MICROSOMAL MIXED FUNCTION OXIDATION IN HUMAN FETAL TISSUES

Knowledge of fetal drug metabolism is a necessary prerequisite for determining the safe and rational use of drug therapy during pregnancy. Heme synthesis and drug metabolism were studied in 40 human fetuses aborted by hysterotomy and prostaglandins. Δ aminolevulinic acid synthetase (ALAS), the rate limiting enzyme in heme synthesis, could be measured in livers and adrenals of fetuses aborted by hysterotomy but not in fetuses aborted by prostaglandins. Mean levels of ALAS ± SE were 74.3 ± 6.4 mμmol/gm liver in 5 fetuses aborted by hysterotomy. Aryl hydrocarbon hydroxylase activity (AHH) was highest in the adrenal gland and progressively lower in liver, intestine, testes, kidney and lung in fetuses from both groups. AHH activity in the adrenal gland was positively correlated with the concentration of microsomal cytochrome P-448 and negatively correlated with the concentration of cytochrome P-420. Secobarbital when added to human fetal liver and adrenal microsomes resulted in a type II difference spectrum (peak 430 nm, trough 406 nm) although when added to microsomes from chick embryos a type I spectrum (peak 385 nm, trough 419 nm) resulted. These studies 1) supply the first reported measurements of ALAS in human fetal tissues, 2) show that fetuses aborted by prosta-glandins are useful for studies of AHH activity, 3) provide evidence that AHH in the fetal adrenal is mediated by cytochrome P-448 and 4) show species differences in substrate binding to embryonic microsomal proteins.

Expression of aryl hydrocarbon hydroxylase induction in mouse tissues in vivo and in organ culture

The elevation of aryl hydrocarbon hydroxylase by various microsomal enzyme inducers in mouse tissues from five inbred strains was examined in vivo and in fetal liver expiants. The magnitude of 3-methylcholanthrene- or β-naphthoflavone-inducible AHH activities in the intact animal varied greatly with the tissue and strain—from no induction in the liver and less than a 2- to 3-fold increase in the lung of DBA/2+ and AKR mice to 4- to 5- and 6- to 7-fold elevation, respectively, in the liver and lung of C57BL mice. Treatment of At or C3H + mice with these inducers increased AHH activity in liver and lung to levels which were intermediate between those observed with tissues from DBA/2+ and C57BL mice. These strain-specific differences in the expression of AHH induction in response to polycyclic hydrocarbons and flavones were also present in fetal liver expiants and were measurable as early as 6 days before parturition. In expiants derived from polycyclic hydrocarbon-“responsive” strains, the extent of enzyme induction was greatest with 4′-bromoflavone, less with β-naphthoflavone and least with 3-methylcholanthrene. Trans-1, 2-dihydroxy-3-methylcholanthrene was about twice as effective in this regard as the parent compound 3-methylcholanthrene. Among expiants from 3-methylcholanthrene-“resistant” strains (DBA/2+, AKR), a disparity in the effects of different classes of compounds was apparent: the flavone derivatives induced aryl hydrocarbon hydroxylase activity from DBA/2+ and AKR expiants by 2- to 3-fold despite the absence of polycyclic hydrocarbon induction in these cultures. Furthermore, although phenobarbital was a comparatively weak inducer under the conditions used in these experiments, this substance stimulated aryl hydrocarbon hydroxylase activity from 3-methylcholanthrene-“responsive” and -“resistant” explants by similar degrees (i.e., about 30%). The results are discussed in the light of previous suggestions on the genetically determined regulation of aryl hydrocarbon hydroxylase induction in mouse tissues.