Cytochrome P450 aromatase expression in human seminoma

Background  

The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis.     

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis

Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).     

Cytochrome P450

Since 1993, three new cytochrome P450 X-ray structures have been determined, giving a total of four known structures. Two of the new structures are in the substrate-free form and one is substrate-bound. These new structures, together with a wealth of mutagenesis studies on various P450s, have provided considerable information on what structural features control substrate specificity in P450s. In addition, some important insights into the catalytic mechanism have been made.     

植物细胞色素P450

对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。     

Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses

The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450arom) enzyme that converts C19 androgens to C18 estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the breeding and non-breeding seasons. Tissue was fixed and prepared for immunocytochemistry (ICC), carried out with an antiserum against human placental P450arom. In prepubertal colts, there was distinct immunopositive staining of a similar degree within both the Leydig cell and the seminiferous tubule. Horses in the pubertal group had strong Leydig cell immunopositive staining and a slight degree of positive staining within the seminiferous tubules. Postpubertal stallions exhibited definitive immunopositive staining within Leydig cells but not within the seminiferous tubules. Therefore, P450arom is present within the Leydig cell throughout sexual development. In contrast, the presence of P450arom within the seminiferous tubule based upon ICC appeared to be gone by adulthood, suggesting that an age-dependent shift in the locale of this enzyme as the stallion matures.     

Cytochrome P450 biosensors-a review

Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice.     

Cytochrome P450s and chemoprevention

The cytochrome P450 mono-oxygenase system represents a major defence against chemical challenge from the environment, constituting part of an adaptive response mounted by an organism following exposure to harmful agents. Cytochrome P450s are also able to catalyse the activation of compounds to toxic products, and participate in a variety of essential 'housekeeping' functions, such as biosynthesis of steroid hormones and fatty acid oxidation. It is clear that the modulation of expression of these enzymes can have a significant effect on chemical toxicity, carcinogenicity and mutagenicity. The concept of cancer chemoprevention, i.e. the administration of a (non-toxic) chemical or dietary component in order to prevent neoplastic disease or to inhibit its progression, is an attractive one. Despite this, relatively little work has been done to characterize the ability of putative chemopreventive agents to modulate P450 expression, or to understand the interaction between P450s and chemopreventive agents. Before chemopreventive treatment can become a reality, it is essential that this complex issue is addressed; for instance, it is likely that any single chemopreventive agent will induce more than one P450 isoenzyme, and while altered expression of a particular P450 may attenuate the effects of one toxic agent, the effects of others might well be potentiated. Our laboratory has created a transgenic mouse line in which the rat CYP1A1 promoter drives expression of the beta-galactosidase gene. These mice can be used to define which compounds act via the Ah receptor, in which tissues, and at which stage of development. We are currently developing another mouse line in which beta1-galactosidase expression is controlled by the mouse GstA1 promoter, allowing us to define the role of the antioxidant responsive element in the action of chemopreventive agents. Finally, using cre-loxP transgenic technology, we have generated a mouse line in which P450 reductase can be deleted in a conditional, i.e. tissue-specific, manner, permitting us to investigate the role of P450s in chemoprevention in a more defined manner.     

细胞色素P450与肿瘤

本文综棕了细胞色素Ｐ４５０同工酶与致癌物代谢、与抗癌药的相互作用以及化的关系,并对调控Ｐ４５０同工酶以防治肿瘤的策略进行了论述。由于Ｐ４５０同工酶具有多态性、工物特异性及可诱导性的特点,在调控Ｐ４５０同工酶以防治肿瘤的问题上,针对不同人群、不同疾病状况及不同用药方案可能需采取抑制或诱导的不同策略。     

Cytochrome P450 in adrenocortical mitochondria

Summary Cytochrome P₄₅₀ in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P₄₅₀ each catalyzing a specified reaction have been purified to a homogeneous state, that is, P_450scc having cholesterol side chain cleavage activity and P₄₅₀₁₁ having 11-hydroxylation activity. The properties of these purified P₄₅₀'s as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P₄₅₀ in the mitochondria) preparation in situ.Among many findings, both purified cytochromes P₄₅₀ were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P₄₅₀. These effects are important from the point of view that the primary role of the heme of cytochrome P₄₅₀ is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P₄₅₀ in the reconstituted system have also been described Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.     

细胞色素P450与除草剂代谢

细胞色素P450是广泛存在于生物中的一类具有混合功能的血红素氧化酶。P450对除草剂代谢的机制及反应类型是多样的,与除草剂代谢相关的P450基因的植物转基因研究得到了具有不同除草剂抗性的转基因植物。文章就这方面的研究进展作介绍。     

Plant Cytochrome P450 Monooxygenases

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated.     

微生物细胞色素P450与胆固醇的生物代谢

细胞色素P450(CYP450)是一类含亚铁血红素的单加氧酶,广泛存在于各类生物体内,参与多种外源物质的代谢和内源物质的转化,如甾类激素、胆汁酸、胆固醇等的代谢。胆固醇是一种环戊烷多氢菲的衍生物,也是人类重要的脂类物质和许多特殊生物活性物质的前体之一,当其过量时会导致高胆固醇血症、动脉粥样硬化、静脉血栓生成等,对机体产生不利的影响。微生物CYP450酶可催化胆固醇的生物代谢,特别是其中的CYP125酶是胆固醇分解代谢起始的关键酶,可用作调节胆固醇代谢的药物靶标。     

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.     

细胞色素P450与癌症基因治疗

细胞色素Ｐ45 0基因与癌症化疗潜药(该类潜药可通过Ｐ45 0催化的加单氧酶反应而活化 )结合的癌症基因疗法引人注意[1]一些抗癌药物可被Ｐ45 0酶系代谢 (表1 )。环磷酰胺 (ＣＰＡ )、异环磷酰胺 (ｉｆｏｓ ｆａｍｉｄｅ)、甲基苄肼、甲苄肼 ( ｐｒｏｃａｒｂａｚｉｎｅ)和氮烯咪胺 (ｄａｃａｒｂａｚｉｎｅ) ,经特定Ｐ45 0酶系的代谢所产生的中间物具有抗癌活性。硫代ＴＥＰＡ、阿霉素、依托泊甙 (ｅｔｏｐｏｓｉｄｅ)和三苯氧胺 (ｔａｍｏｘｉｆｅｎ)本身具有抗癌活性 ,但通过Ｐ45 0代谢形成的具有细胞毒性的代谢物 ,抗癌活性可得到加强。还…     

Unusual Cytochrome P450 Enzymes and Reactions

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO³⁺), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function.     

细胞色素P450与植物的次生代谢

细胞色素Ｐ４５０是动植物及微生物体内的一类重要的混合功能的血红素氧化还原酶类，是一个基因超家族。它可催化多种化学反应，在防御生物免受外界侵害方面有重要作用。植物Ｐ４５０广泛参与次生代谢产生有植保素功能的物质和对除草剂、杀虫剂等外毒素的生物转化解毒代谢。许多植物Ｐ４５０已被分离纯化，更多的植物Ｐ４５０的基因被克隆和外源表达。对植物Ｐ４５０的表达调控也取得了一些进展，但在抗虫和抗除草剂的农作物基因工程方面尚在起步阶段。     

Reactive Intermediates in Cytochrome P450 Catalysis

Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450_ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis.