Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

Differential expression of CYP102 in Bacillus megaterium by 17-beta-estradiol and 4-sec-butylphenol

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.     

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.     

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.     

2,3,7,8-tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.     

Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.