Prince K. Zachariah

Further studies on the properties of human placental microsomal cytochrome P-450.

Abstract Spectral studies of human placental microsomal cytochromes provided evidence that androstenedione will bind either to two separate cytochromes or to two sites on the same cytochrome. Scatchard and Lineweaver-Burk plots indicated the presence of a binding site with high affinity and low capacity and a second site with a lower affinity but higher capacity. Both sites were discernible in the presence of high concentrations of NADPH, and similar binding constants were obtained. The absorption maximum (450 nm) of the carbon monoxide complex of NADPH-reduced placental cytochrome P-450 could be abolished completely by additions of low concentrations of androstenedione. 19-hydroxyandrostenedione or 19-oxoandrostenedione, but not by high concentrations of 19-norandrostenedione. 19-nortestosterone, pregnenolone or benzo[a]pyrene. Compounds capable of binding significantly to placental microsomal cytochrome P-450 appeared to fall into three categories: C-19 or C-18 steroids, with structures similar to that of androstenedione or 19-norandrostenedione: C-18 steroids with structures very similar to that of β-estradiol; or substances capable of forming ferrihemochromes. A large number of foreign organic compounds which produced type-I binding spectra in rat liver microsomes exhibited either no or very minimal binding to placental cytochrome P-450. Most compounds that exhibited significant binding to placental cytochrome P-450 also were inhibitors of rates of androstenedione aromatizalion (aromatase activity). Important exceptions were carbon monoxide, metyrapone and nicotinamide. Diethylstilbestrol acted as a relatively potent inhibitor of aromatase activity but displayed no discernible binding. Results of the study strongly supported a functional role for placental cytochrome P-450 in the aromatization reaction.

Transplacental induction of carcinogen-hydroxylating systems with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of a highly toxic herbicide contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) upon rates of hydroxylation of N-2-fluorenylacetamide (FAA) and of benzo[a]-pyrene (BP) were studied in fetal livers, in placentas, maternal livers, and adrenal glands of pregnant Sprague-Dawley rats. Aryl hydrocarbon hydroxylase (AHH) activity was increased 14- and 100-fold in maternal and fetal livers, respectively, but only minimal increases were observed in placentas and adrenal glands. Similar results were obtained with respect to rates of 7-, 5-, N-, 3-, and 1-hydroxylations of FAA. Histologic examinations revealed extensive cellular damage in the placentas as well as pathologic changes in fetal and maternal livers. Electron microscopy indicated enlarged mitochondria and extensive glycogen deposition in maternal and fetal livers and increases in the amounts of rough endoplasmic reticulum in fetal livers. Analyses of BP metabolism with high-pressure liquid chromatography also revealed that the formation of diols (relative to phenols) was markedly increased in fetal and maternal livers following treatments with TCDD. The results suggested that the relative lack of response of fetal hepatic mixed-function oxidase systems to commonly employed inducing agents (polycyclic aromatic hydrocarbons) was not due to a lack of appropriate structural and/or regulatory genes required for the expression of enzyme induction during fetal life.

Inhibition of human placental mixed-function oxidations with carbon monoxide: reversal with monochromatic light.

Abstract The present investigation was designed to further examine the functions of cytochrome(s) P-450 (P-450 hpm ) present in the endoplasmic reticulum of the human placental syncitium. The antagonistic effects of androstenedione and 19-norandrostenedione with regard to respective inhibition and facilitation of the binding of carbon monoxide to the cytochrome(s) were exploited in the examination of functionality. The presence of various concentrations of 19-norandrostenedione in reaction vessels permitted carbon monoxide to inhibit the conversion of androstenedione to estrone in a concentration-dependent fashion. Optimal light-reversal of the inhibition was achieved at a wavelength of 450 nm. Optimal reversal of inhibition of placental microsomal aryl hydrocarbon hydroxylase by carbon monoxide likewise was achieved with light at a wavelength of 450 nm, thus providing definitive evidence for a role for P-450 hpm in placental aryl hydrocarbon hydroxylase (AHH). However, neither androstenedione nor 19-norandrostenedione affected the capacity of carbon monoxide to inhibit placental AHH. The results provided evidence for the hypothesis that at least two species of cytochrome P-450 are present in human placental endoplasmic reticulum. One species, not detectable with light spectroscopy in these experiments, appeared to function in the biotransformation of foreign hydrocarbons. The major physiologic role of the spectrally visible species appeared to be in the biosynthesis of estrogens from endogenous C-19 steroids.

Comparative studies on the oxidation and reduction of drug substrates in human placental versus rat hepatic microsomes.

Abstract Human placental microsomes prepared by conventional methods were compared with analogous preparations from adult, male rat livers with respect to biochemical components and systems which could affect rates of mixed-function oxidation and reduction of drugs and steroids in vitro . Each of the electron transport components required for the mixed-function oxidation of drugs in hepatic systems was present in lower concentrations in placental than hepatic microsomes. In contrast to hepatic microsomes, placental microsomes which exhibited unusually high aryl hydrocarbon hydroxylase activities did not contain increased concentrations of the electron transport components. Evidence was provided to indicate that rapid degradation of initial electron donors (reduced pyridine nucleotides) was not responsible for the observance of low or negligible drug metabolic activities observed in incubations with placental micro-somes. Cytochromes P-450 and b 5 and their corresponding reductases were shown to be present in human placental preparations. NADPH and NADH-dependent cytochrome c reductase activities in placental microsomes were somewhat lower but comparable to those determined in hepatic preparations. However, cytochromes b 5 and P-450 and contaminating hemoglobin and methemoglobin accounted for less than 56 per cent of the total heme present in placental microsomes. Rapid degradation of placental cytochrome P-450 was observed at 37° in the presence of sodium hydrosulfite, but conversion to cytochrome P-420 was minimal after incubation for 1 hr in the presence of NADPH at the same temperature. It was con- sidered probable that the low rates of drug biotransformation observed would be explicable in terms of high substrate specificities of the placental enzymic components.

Analysis of the biotransformation of benzo[a]pyrene in human fetal and placental tissues with high-pressure liquid chromatography☆

Abstract The biotransformation of benzo[a]pyrene (BP) was studied in vitro in the presence of microsomal fractions of placental and fetal tissues from humans and monkeys (Macaca nemestrina) . Metabolites formed in the incubation flasks were extracted and separated by means of high-pressure liquid chromatography utilizing a microparticulate column. In general, the formation of diols and quinones in fetal and placental homogenates was undetectable following 15 -min incubations. The formation of phenolic metabolites, however, was easily measurable in fetal liver and lung and in the placenta but not in fetal spleen, kidney, pancreas or adrenal gland. The latter observation contrasted with high specific activities measured in the fetal adrenal gland with the fluorometric assay for aryl hydrocarbon hydroxylase activity. In placentas from cigarette smokers, relatively large quantities of an unidentified metabolite(s) appeared in metabolic profiles. This metabolite(s) did not co-chromatograph with any of the standard metabolites and the retention time was between those of the 9,10 - and 4,5 -diols. The same placental tissues catalyzed the covalent binding of BP to DNA and were far more active in this regard than any of the other fetal tissues investigated. The data indicated a correlation between metabolic profiles and capacity for catalyzing covalent binding to DNA for fetal/placental tissues.

Interactions of steroids with human placental cytochrome P-450 in the presence of carbon monoxide.

Abstract Spectral analyses of the carbon monoxide (CO) complex of human placental microsomal cytochrome P-450 revealed absorption maxima at 426 and 450 nm when NADPH (2×10 −4 M) was utilized as a reducing agent. Additional NADPH or NADH did not produce any further increases in the absorption maximum at 450 nm. A period of 10–15 minutes was required for the complete reduction. Various steroids were added to both sample and reference cuvettes to examine their interactions with the CO-cytochrome P-450 complex. The resulting spectral changes indicated that low concentrations of steroids (≃10 −7 M) such as androstenedione, 19-hydroxyandrostenedione, 19-oxoandrostenedione and testosterone completely eliminated the absorbance maxima at 450 nm while 19-norandrostenedione, 19-nortestosterone, pregnenolone and benzo[a]-pyrene did not eliminate this peak. Since ample time was allowed to reduce the cytochrome P-450 with NADPH, the observed interaction of steroids with cytochrome P-450 in the presence of CO does not represent an effect on reductase activity, but on the formation of the CO-cytochrome P-450 complex.

Displacement of carbon monoxide from placental cytochrome P-450 by steroids: Antagonistic effects of androstenedione and 19-norandrostenedione

Summary Displacement of bound carbon monoxide (CO) from NADPH- or NADHreduced human placental microsomal cytochrome P-450 by androstenedione was reversed by 19-norandrostenedione or 19-nortestosterone. Conversely, the capacity of the 19-norsteroids to augment the formation of the CO-cytochrome complex was antagonized by androstenedione. The effects produced by each of the steroids investigated were independent of the sequence of additions of reduced pyrioine nucleotides, steroids and CO, suggesting that the observed results were not due to effects on rates of enzymic reduction of cytochrome.

Spectral characteristics of human fetal adrenal microsomes

Abstract Investigations of human fetal adrenal gland microsomes indicated that a carbon monoxide binding pigment had an absorption maximum of 446 to 448 nm. This pigment, upon heat treatment at 37°C was degraded to the form of cytochrome p-420. NADPH reduced cytochrome p-450 slowly and completely. Typical concentrations of 0.75 and 0.16 nmoles/mg protein cytochrome P-450 and b 5 , respectively, were observed. Reduced ethylisocyanide spectra were similar to those of rat hepatic microsomes with absorption maxima at 430 as well as 454 nm. Typical type I spectral changes were observed with progesterone, 17-α-OH-progesterone, pregnenolone and androstenedione when these steroids were added to the sample cuvettes. Androstenedione exhibited an apparent spectral dissociation constant (K S ) of 5×10 −6 M pregnenolone and progesterone exhibited higher affinities with apparent dissociation constants of 1.1×10 −7 M and 1.8×10 −7 M, respectively. The maximal absorbance change induced by androstenedione was lower (Emax = 0.027 per mg protien) than the changes in absorbance maxima induced by pregnenolone or progesterone (Emax = 0.060 and 0.047 per mg protein, respectively) when saturating concentrations of these steroids were added to the sample cuvettes. Ethylmorphine and aminopyrine (10 −3 M final concentrations) did not exhibit observable spectral changes; however, type II spectra could be elicited with aniline and nicotinamide and apparent dissociation constants of 3.5×10 −2 M and 2.5×10 −2 M, respectively, were obtained.

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione

The aromatization of androst-4-en-3,17-dione or 17beta hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17beta-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the ce titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.

Interactions of various 19-nor steroids with human placental microsomal cytochrome P-450 (P-450hpm).

Each of seven 19-nor steroids exhibited the capacity to facilitate the binding of carbon monoxide (CO) to human placental microsomal cytochrome P-450 although quantitative differences were shown to exist. In every case the facilitation was antagonized by androstenedione. 19-Norandrostenedione produced the most pronounced effect followed by 19-nortestosterone, nandrolone decanoate, norethandrolone, norgestrel, norethynodrel and norethindrone in that order. All steroids investigated produced typical type I binding spectra when added to placental microsomes. Scatched plots also indicated binding of each steroid to two sites--a high-affinity, low-capacity binding site and a low-affinity, high-capacity binding site. Correlations between affinity for either site and capacity to facilitate binding of CO to the cytochrome were not observed nor were there good correlations between maximal absorbance differences (approximately390-420 nm) producible and facilitation capacity. It was therefore concluded that no definitive relationships existed between facilitation capacity and qualitative or quantitative aspects of the steroid-binding spectra. The capacity to facilitate CO binding appeared to reside in the absence of a chemical group substituted at the 10 position on molecules of androgenic steroids since all investigated steroids possessing 10-methyl or other 10-substituted groups either had no effect on the CO-binding spectrum or caused a displacement of CO from ferrous heme. In contrast, all steroids studied that lacked a substitution at C-10 (19-nor steroids) produced a facilitating effect on heme-ligand binding.