Robert E. Kramer

Dose-dependent actions of thyroxine on hepatic drug metabolism in male and female rats

Abstract Studies were carried out to compare the effects of various doses of thyroxine (T 4 ) on hepatic drug metabolism in male and female rats and to evaluate the role of the pituitary gland in the modulation of T 4 action. Administration of small amounts of T 4 (2.5 to 5 μg/100g body wt/day) to hypophysectomized rats of either sex increased hepatic ethylmorphine demethylase, benzo(a)pyrene hydroxylase and aniline hydroxylase activities. Larger amounts of T 4 (12.5 to 50 μg) reversed the stimulatory effects of the smaller doses. T 4 treatment produced dose-dependent decreases in hepatic cytochrome P-450 content and increases in NADPH-cytochrome c reductase activity in hypophysectomized rats of both sexes. Qualitatively similar effects were produced by T 4 administration to thyroidectomized male and female rats. However, larger doses of T 4 were required for maximum stimulation of drug metabolism in thyroidectomized than in hypophysectomized animals. The results indicate that physiological amounts of T 4 Uniformly stimulate hepatic drug metabolism in both male and female rats. Supraphysiological amounts, however, inhibit metabolism of some substrates and produce sex differences in T 4 actions. The effects of T 4 are demonstrable in the absence of the pituitary gland but pituitary-dependent factors appear to modulate the magnitude of the response to T 4 .

Differential control of adrenal drug and steroid metabolism in the guinea pig.

Abstract Studies were carried out to compare the effects of several physiological variables on adrenal microsomal drug (ethylmorphine demethylation) and steroid (21-hydroxylation) metabolism in guinea pigs. The rate of adrenal ethylmorphine (EM) metabolism increased with maturation in males but not females, resulting in a sex difference (M > F) in adrenal enzyme activity in adult guinea pigs. Twenty-one hydroxylase activity, in contrast, was similar in adrenals from males and females. The concentration of adrenal microsomal cytochrome P-450 was unaffected by age or sex. ACTH administration decreased adrenal EM demethylase activity but did not affect 21-hydroxylation. Testosterone, when given to female guinea pigs, increased the rate of EM metabolism and decreased 21-hydroxylase activity. Various compounds known to interact with adrenal microsomal cytochrome P-450 had divergent effects on EM metabolism and 21-hydroxylation in vitro . Prostaglandins E1 and F2α, spironolactone, and canrenone inhibited EM demethylation but not 21-hydroxylation. Simple aromatic hydrocarbons (benzene, toluene), in contrast, inhibited 21-hydroxylation but did not affect EM metabolism. The results indicate that adrenal drug and steroid metabolism are independently regulated and that different terminal oxidases (cytochrome P-450) are probably involved in adrenal 21-hydroxylation and EM demethylation.

Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 in the guinea pig.

Abstract Addition of simple aromatic hydrocarbons (benzene, ethylbenzene, naphthalene) to guinea pig adrenal microsomes produced typical Type I difference spectra (ΔOD 385-420 ). Spectral dissociation constants ( K s ) for each indicated a far higher affinity for adrenal than hepatic cytochrome P-450. Hydrocarbon affinities for adrenal cytochrome P-450 were similar to that for progesterone, an endogenous steroid substrate. Ethylmorphine and aniline produced Type I and Type II spectral changes respectively in adrenal microsomes. The K s , and magnitude of spectrum for each in adrenals was similar to that in livers. Nonetheless, demethylation of ethylmorphine proceeded far more rapidly in adrenal than hepatic tissue. The Michaelis constants ( K m ) for ethylmorphine metabolism in both tissues were similar. Although the aniline-induced difference spectra in adrenal and hepatic microsomes did not differ substantially, aniline hydroxylase activity was far greater in liver. Pretreatment of guinea pigs with phenobarbital or 3-methylcholanthrene increased hepatic but not adrenal ethylmorphine metabolism. Spironolactone pretreatment, in contrast, did not affect hepatic metabolism, but significantly lowered adrenal demethylase activity. The results indicate a relative non-specificity of guinea pig adrenal microsomal cytochrome P-450 and suggest that the adrenal cortex may represent a significant site for the extra-hepatic metabolism of foreign compounds in the guinea pig.

Divergence of growth hormone actions on hepatic drug metabolism in the presence and absence of the pituitary gland

Abstract Experiments were carried out to compare the effects of growth hormone on hepatic drug oxidation in normal and hypophysectomized rats. Administration of growth hormone to normal male rats lowered hepatic microsomal cytochrome P-450 content and decreased the rates of ethylmorphine n-demethylation and aniline hydroxylation. These effects were fully manifested in orchiectomized or adrenalectomized males, excluding a dependence upon endogenous steroids. Growth hormone was without effect on hepatic drug metabolism or cytochrome P-450 content in normal female rats. In contrast to its actions in animals with intact pituitary glands, administration of growth hormone to hypophysectomized rats of either sex increased the rate of ethylmorphine metabolism. Furthermore, in both males and females, aniline hydroxylation and microsomal cytochrome P-450 content were unaffected by growth hormone in the absence of the pituitary gland. Prolactin administration did not affect hypophysectomized or in normal rats of either sex. The results indicate that the nature of growth hormone actions on hepatic drug oxidation is pituitary-dependent and probably intertwined with the effects of other hormones. Furthermore, the direct physiological effects of growth hormone on hepatic mixed function oxidases seem to depend upon the substrate employed.

Interaction of metyrapone with adrenal microsomal cytochrome P450 in the guinea pig

Abstract Studies were carried out to compare the actions of metyrapone on adrenal mitochondrial and microsomal cytochrome P450-containing enzymes in the guinea pig and rat. As expected. addition of metyrapone to adrenal mitochondria inhibited 11β-hydroxylation in both species. the shape of the type II difference spectrum produced by metyrapone in mitochondria differed somewhat in rat ( g DO.D. 425−405 nm ) and guinea pig ( g DO.D. 425−390 nm ) and the magnitude of the speetrum was far greater in rat adrenal mitochondria, paralleling species differences in cytochrome P450 concentration (rat > guinea pig). In rat adrenal microsomes, metyrapone produced a small “reverse type I” spectral change (ΔO.D. 420-385nm ) but did not affect either 21-hydroxylation or the interaction of progesterone with eytochrome P450(as determined spectrally). In guinea pig adrenal microsomes, in contrast, metyrapone produced a large type II spectral change (ΔO.D. 423-408nm ) and inhibited both 21-hydroxylation and ethylmorphine demethylation, cytochrome P450-dependent reactions. The magnitudes of type I spectra produced by 17α-hydroxyprogesterone and ethylmorphine in guinea pig adrenal microsomes were significantly diminished by prior addition of metyrapone. The results indicate that metyrapone interacts with both microsomal and mitochondrial cytochrome P450 in the guinea pig and that its adrenal sites of action, therefore, are species dependent.

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.

Relation of the pituitary gland to gonadal hormone effects on the adrenocortical 11β-hydroxylase system in rats

Studies were conducted to further examine the mechanisms responsible for gonadal hormone effects on the rat adrenocortical 11beta-hydroxylase system. Despite higher concentrations of cytochrome P-450 and larger 11-deoxycorticosterone (DOC)-induced difference spectra in adrenal mitochondria from females than males, no sex difference in 11beta-hydroxylase activity was observed. The pregnenolone-induced difference spectrum, indicative of cholesterol binding to cytochrome P-450, also was similar in males and females. Testosterone administration to castrated males lowered both 11beta-hydroxylase activity and mitochondrial cytochrome P-450 content. Estradiol produced the opposite effects in castrated females. However, when given to ACTH-replaced hypophysectomized rats, neither testosterone nor estradiol affected cytochrome P-450 levels or the rate of 11beta-hydroxylation. These observations, taken with the known effects of estradiol and testosterone on ACTH secretion in rats and the effects of ACTH on 11beta-hydroxylation, indicate that gonadal hormone effects on the 11beta-hydroxylase system are mediated by ACTH.