Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.

Effects of cigarette smoking and exposure to cadmium and lead on phenotypic variability of hepatic CYP2A6 and renal function biomarkers in men

Effects of cigarette smoking and exposure to dietary cadmium (Cd) and lead (Pb) on urinary biomarkers of renal function and phenotypic variability of cytochrome P450 2A6 (CYP2A6) were investigated in a group of 96 healthy Thai men with mean age of 36.7 year (19-57 years). In non-smokers, Cd burden increased with age (r = 0.47, P < 0.001). In current smokers, Cd burden increased with both age (r = 0.45, P = 0.01) and number of cigarettes smoked per day (r = 0.32, P = 0.05). Cd-linked renal tubular dysfunction was seen in both smokers and non-smokers, but Pb-linked glomerular dysfunction was seen in smokers only, possibly due to more recent exposure to high levels of Cd and Pb, as reflected by 30-50% higher serum Cd and Pb levels in smokers than non-smokers (P < 0.05). Exposure to dietary Cd and Pb appeared to be associated with mild tubular dysfunction whereas dietary exposure plus cigarette smoking was associated with tubular plus glomerular dysfunction. Hepatic CYP2A6 activity in non-smokers showed a positive association with Cd burden (adjusted P = 0.38, P = 0.006), but it showed an inverse correlation with Pb (adjusted beta = -0.29, P = 0.003), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. In contrast, CYP2A6 activity in current smokers did not correlate with Cd or Pb, but it showed a positive correlation with serum ferritin levels (r = 0.45, P = 0.01). These finding suggest that Pb concentrations in the liver probably were too low to inhibit hepatic synthesis of heme and CYP2A6 and that the concurrent induction of hepatic CYP2A6 and ferritin was probably due to cigarette smoke constituents other than the Cd and Pb. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Evidence for Concurrent Effects of Exposure to Environmental Cadmium and Lead on Hepatic CYP2A6 Phenotype and Renal Function Biomarkers in Non Smokers

We examined the interrelationships between phenotype of hepatic cytochrome P450 2A6 (CYP2A6), nephropathy, and exposure to cadmium and lead in a group of 118 healthy Thai men and women who had never smoked. Their urinary Cd excretion ranged from 0.05 to 2.36 mug/g creatinine, whereas their urinary Pb excretion ranged from 0.1 to 12 mug/g creatinine. Average age and Cd burden of women and men did not differ. Women, however, on average showed a 46% higher urinary Pb excretion (p < 0.001) and lower zinc status, suggested by lower average serum Zn and urinary Zn excretion compared with those in men. Cd-linked nephropathy was detected in both men and women. However, Pb-linked nephropathy was seen only in women, possibly because of higher Pb burden coupled with lower protective factors, notably of Zn (P < 0.001), in women compared with men. In men, Pb burden showed a negative association with CYP2A6 activity (adjusted beta = -0.29, p = 0.003), whereas Cd burden showed a positive association with CYP2A6 activity (adjusted beta = 0.38, p = 0.001), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. The weaker correlation between Cd burden CYP2A6 activity in women despite similarity in Cd burden between men and women is consistent with opposing effects of Pb and Cd on hepatic CYP2A6 phenotypic expression. A positive correlation between Cd-linked nephropathy (urinary N-acetyl-beta-D-glucosaminidase excretion) and CYP2A6 activity in men (r = 0.39, p = 0.002) and women (r = 0.37, p = 0.001) suggests that Cd induction of hepatic CYP2A6 expression and Cd-linked nephropathy occurred simultaneously.

Acute cadmium chloride administration induces hepatic and renal cytochrome P450 2A5 in the mouse

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 Amol/kg body weight, i.p.) of cadmium chloride (CdCl2) at various time points. The total CYP content of kidney microsomes started to decrease 4 hours earlier than in the liver (P < 0.05), with maximal decreases at 24 hours of 56% and 85% in the liver and kidney, respectively. In contrast, both hepatic and renal coumarin 7-hydroxylase (COH) activity (indicative of CYP2A5 activity) relative to total CYP content started to progressively increase at 8 hours, with renal activity 61 times higher than the hepatic activity. Maximum increases were observed, 15-fold in the liver and 64-fold in the kidney after 24 hours. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 hours after treatment, respectively and decreased to almost half 6 hours later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 hours. This study demonstrates that hepatic and renal CYP2A5 is upregulated by cadmium with a faster response in the kidney than in the liver. This observation is concordant with the fact that kidney is the target organ for cadmium toxicity. The observed increase in the mRNA but not in protein levels after maximal induction suggests involvement of post-transcriptional mechanisms in the regulation of CYP2A5 expression by cadmium.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) - a breakdown product of haem with cytoprotective and toxic properties - is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5-10 h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC50 approximately equal to the substrate concentration. The 7-methoxyresorufin 7-O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis.

Vascular biology of magnesium and its transporters in hypertension

Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on myriad biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoconstrictor agents. Mammalian cells regulate Mg(2+) concentration through special transport systems that have only recently been characterized. Magnesium efflux occurs via Na(2+)-dependent and Na(2+)-independent pathways. Mg(2+) influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A2, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg(2+) deficiency in hypertension and other cardiovascular pathologies. In particular, increased Mg(2+) efflux through dysregulation of the vascular Na(+)/Mg(2+) exchanger and decreased Mg(2+) influx due to defective vascular and renal TRPM6/7 expression/activity may be important in altered vasomotor tone and consequently in blood pressure regulation. The present review discusses the role of Mg(2+) in vascular biology and implications in hypertension and focuses on the putative transport systems that control magnesium homeostasis in the vascular system. Much research is still needed to clarify the exact mechanisms of cardiovascular Mg(2+) regulation and the implications of aberrant cellular Mg(2+) transport and altered cation status in the pathogenesis of hypertension and other cardiovascular diseases.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

Practical Quality Control: Comparision of Methods on the Quantification of Stationary Phases in Paper and Thin-Layer Chromatographic Systems

Introduction: Paper and thin layer chromatography methods are frequently used in Classic Nuclear Medicine for the determination of radiochemical purity (RCP) on radiopharmaceutical preparations. An aliquot of the radiopharmaceutical to be tested is spotted at the origin of a chromatographic strip (stationary phase), which in turn is placed in a chromatographic chamber in order to separate and quantify radiochemical species present in the radiopharmaceutical preparation. There are several methods for the RCP measurement, based on the use of equipment as dose calibrators, well scintillation counters, radiochromatografic scanners and gamma cameras. The purpose of this study was to compare these quantification methods for the determination of RCP. Material and Methods: 99mTc-Tetrofosmin and 99mTc-HDP are the radiopharmaceuticals chosen to serve as the basis for this study. For the determination of RCP of 99mTc-Tetrofosmin we used ITLC-SG (2.5 x 10 cm) and 2-butanone (99mTc-tetrofosmin Rf = 0.55, 99mTcO4- Rf = 1.0, other labeled impurities 99mTc-RH RF = 0.0). For the determination of RCP of 99mTc-HDP, Whatman 31ET and acetone was used (99mTc-HDP Rf = 0.0, 99mTcO4- Rf = 1.0, other labeled impurities RF = 0.0). After the development of the solvent front, the strips were allowed to dry and then imaged on the gamma camera (256x256 matrix; zoom 2; LEHR parallel-hole collimator; 5-minute image) and on the radiochromatogram scanner. Then, strips were cut in Rf 0.8 in the case of 99mTc-tetrofosmin and Rf 0.5 in the case of 99mTc-HDP. The resultant pieces were smashed in an assay tube (to minimize the effect of counting geometry) and counted in the dose calibrator and in the well scintillation counter (during 1 minute). The RCP was calculated using the formula: % 99mTc-Complex = [(99mTc-Complex) / (Total amount of 99mTc-labeled species)] x 100. Statistical analysis was done using the test of hypotheses for the difference between means in independent samples. Results:The gamma camera based method demonstrated higher operator-dependency (especially concerning the drawing of the ROIs) and the measures obtained using the dose calibrator are very sensitive to the amount of activity spotted in the chromatographic strip, so the use of a minimum of 3.7 MBq activity is essential to minimize quantification errors. Radiochromatographic scanner and well scintillation counter showed concordant results and demonstrated the higher level of precision. Conclusions: Radiochromatographic scanners and well scintillation counters based methods demonstrate to be the most accurate and less operator-dependant methods.

Patients with chronic hepatitis C and normal transaminases

Hepatitis C virus infection evolves progressively persisting in the majority of patients (85%). Most patients have high ALT (alanine aminotransferase) levels and approximately 25% normal ALT. The latter are usually female and there is no association between genotype and severity of hepatic lesion. Histologic analysis usually shows small lesion and absence or low amount of fibrosis, despite cirrhosis having been reported. Aiming at assessing prevalence, demographic, genotypical and anatomopathological characteristics in patients with normal ALT levels, we have carried out a study of 68 chronic hepatitis C patients between January 1997 and April 2000. There was a prevalence of 13.8% chronic hepatitis C patients with normal ALT levels, 45.6% of which were male and 54.4% female, the mean age being 38 +/- 13 years. We found a predominance of genotype 1 in 84.7% of the patients, genotype 2 in 6.8% and genotype 3 in 10.7%. In 52.9% of the cases liver biopsies revealed liver reaction, periportal activity score 0-1 was observed in 85.3% of the patients and score 2-4 was seen in 14.7%. Structural activity score 0-1 was seen in 73.5% of the patients and score 2-4 in 26.5% of them. Periportal activity > 2 and structural activity > 1 was seen in 29%, but steatosis was not seen in 73.5%. Our results suggest the need to revisit for liver biopsy practice in patients with Chronic Hepatitis C and normal transaminases.

Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas

Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ß-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ß-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates.

Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.

Ethanol consumption and pineal melatonin daily profile in rats

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in beta 1 and alpha 1 adrenergic receptors` mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Mineral Trioxide Aggregate-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise estrutural e funcional da interação física entre eIF5A e suas enzimas modificadoras em Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)