Screening of the aflatoxin M1 metabolite in urine samples of residents in Terengganu, Malaysia.

A study was conducted to screen the occurrence and level of aflatoxin M1 (AFM1) in urine samples of 206 urban and rural residents in Terengganu, Malaysia. The level of AFM1 was quantified by competitive enzyme-linked immune-absorbent assay (ELISA). Of the 206 samples, 84 were positive for AFM1 (40.8%) in a range of 0.07-5.53 ng/ml (mean = 0.589 ng/ml). Residents of Terengganu are moderately exposed to AFM1. Age, ethnicity, marital status and employment status were associated with urinary level of AFM1. Subjects aged 30 years and above, non-Malays, married, and those unemployed had significantly higher levels of urinary AFM1 (p < 0.05). Since aflatoxin is recognised as a potent-carcinogen for liver cancer and a continuous exposure to this toxin can be fatal, the present findings could provide a baseline for future studies where larger samples and more advanced techniques might be used to find the possible effects of the exposure of this toxin on the community's health.

Global metabolite profiles of rice brown planthopper-resistant traits reveal potential secondary metabolites for both constitutive and inducible defenses.

INTRODUCTION: Brown planthopper (BPH) is a phloem feeding insect that causes annual disease outbreaks, called hopper burn in many countries throughout Asia, resulting in severe damage to rice production. Currently, mechanistic understanding of BPH resistance in rice plant is limited, which has caused slow progression on developing effective rice varieties as well as effective farming practices against BPH infestation. OBJECTIVE: To reveal rice metabolic responses during 8 days of BPH attack, this study examined polar metabolome extracts of BPH-susceptible (KD) and its BPH-resistant isogenic line (IL308) rice leaves. METHODS: Ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) was combined with multi-block PCA to analyze potential metabolites in response to BPH attack. RESULTS: This multivariate statistical model revealed different metabolic response patterns between the BPH-susceptible and BPH-resistant varieties during BPH infestation. The metabolite responses of the resistant IL308 variety occurred on Day 1, which was significantly earlier than those of the susceptible KD variety which showed an induced response by Days 4 and 8. BPH infestation caused metabolic perturbations in purine, phenylpropanoid, flavonoid, and terpenoid pathways. While found in both susceptible and resistant rice varieties, schaftoside (1.8 fold), iso-schaftoside (1.7 fold), rhoifolin (3.4 fold) and apigenin 6-C-α-L-arabinoside-8-C-ß-L-arabinoside levels (1.6 fold) were significantly increased in the resistant variety by Day 1 post-infestation. 20-hydroxyecdysone acetate (2.5 fold) and dicaffeoylquinic acid (4.7 fold) levels were considerably higher in the resistant rice variety than those in the susceptible variety, both before and after infestation, suggesting that these secondary metabolites play important roles in inducible and constitutive defenses against the BPH infestation. CONCLUSIONS: These potential secondary metabolites will be useful as metabolite markers and/or bioactive compounds for effective and durable approaches to address the BPH problem.

The effect of tributyrin supplementation to milk replacer on plasma glucagon-like peptide 2 concentrations in pre-weaning calves.

The objective of this study was to evaluate the effect of tributyrin (TB) supplementation to milk replacer (MR) on performance, health, and blood concentrations of metabolite and glucagon-like peptide (GLP-2) in pre-weaning calves. Twenty Holstein heifer calves were raised on an intensified nursing program using MR supplemented with either palm oil (CON) or TB (TB) at 0.3% (as fed basis) for 7 weeks starting 1 week after birth. Calves were fed a calf starter and kleingrass from the beginning of the study. Blood samples were obtained weekly to measure blood glucose, serum ß-hydroxybutyric acid (BHBA), insulin-like growth factor 1 (IGF-1), and plasma GLP-2 concentrations. Starter DMI and metabolizable energy (ME) intake were lower in TB calves at 46, 47, from 49 to 55 days after birth compared with the CON calves. However, any growth parameters were not affected by TB treatment. Blood glucose, serum BHBA, and IGF-1 concentrations were not affected by TB supplementation. On the other hand, mean plasma GLP-2 concentration among whole experimental period was higher for TB (0.60 ng/ml) compared with CON (0.41 ng/ml). In conclusion, feeding MR supplemented with TB increases plasma GLP-2 concentration, which might counterbalance the growth performance of TB calves despite the decreased ME intake.

Tacrolimus Metabolite M-III May Have Nephrotoxic and Myelotoxic Effects and Increase the Incidence of Infections in Kidney Transplant Recipients.

BACKGROUND: Tacrolimus (Tac) is one of the most commonly used immunosuppressive drugs after solid organ transplantation. Eight Tac metabolites have been described, but their clinical importance remains unclear. The aim of this study was quantification of the 2 major Tac metabolites, 13-O-demethyl (M-I) and 15-O-demethyl (M-III), in kidney transplant recipients and to link them with parameters of kidney and liver function, peripheral blood cell counts, and infection incidence. METHODS: In 81 kidney transplant recipients, concentrations of Tac, M-I, and M-III were measured with the use of liquid chromatography combined with tandem mass spectrometry (LC-MS-MS). RESULTS: There was a negative correlation between M-III levels and estimated glomerular filtration rate (eGFR; r = -0.244; P < .05). Also, a negative correlation between M-III concentrations and red blood cell count (RBC) was found (r = -0.349; P < .05). Neither concentrations of Tac nor of M-I correlated with eGFR or RBC. M-I, M-III, and Tac were not related to alanine aminotransferase activity. Significantly higher Tac and M-III concentrations in the group with all types of infections in comparison with the group without infections were observed (6.95 ± 2.09 ng/mL vs 5.73 ± 2.43 ng/mL [P = .03] and 0.27 ± 0.17 ng/mL vs 0.20 ± 0.11 ng/mL [P = .04], respectively). CONCLUSIONS: The results suggest that higher concentrations of M-III may have a nephrotoxic or myelotoxic effect and result in higher incidence of infections. Further studies are needed to confirm if monitoring of M-III could minimalize adverse effects such as nephrotoxicity or infections.

Effect of two antiandrogens as protectors of prostate and brain in a Huntington's animal model.

The purpose of this work is to know the effect of flutamide and a novel synthetic steroid 3ß-p-Iodobenzoyloxypregnan-4,16- diene-6,20-dione (IBP) on the levels of dopamine, 5-HIAA (5-hydroxyindole acetic acid), and some oxidative stress markers in animal model with Huntington disease. Thirty male Wistar rats divided in groups of 6 animals each were subjected to the following treatment: group A, 3-nitro propionic acid (3-NPA, as inducer of Huntington); group B, flutamide; group C, 3-NPA + flutamide; group D, IBP; and group E, 3-NPA + IBP. Treatment scheme for all groups were at 4 mg/kg/day administered intraperitoneally. The measurement of haemoglobin was carried out from blood while the concentrations of ATPase, 5α-reductase, reduced glutathione (GSH), calcium, H2O2, 5-HIAA, and dopamine were determined from brain and prostate tissues using validated methods. The results depicted a significant decrease of dopamine and GSH in cerebellum/Medulla oblongata of animals treated with IBP. The prostate gland of the same group of treatment also showed a significant decrease in the concentrations of TBARS, H2O2, and total ATPase. In hemispheres of groups D and E, dopamine, H2O2, and total ATPase decreased significantly while in prostate, hemispheres, and cerebellum/Medulla oblongata of groups B and C; calcium, 5α-reductase, ATPase, H2O2, and TBARS were found to witness a significant decrease. Results showed an antiandrogenic activity of flutamide, while the novel steroid IBP showed neuroprotective properties by changes on oxidative stress biomarkers as critical pathways leading to prostate and brain degeneration. Probably steroid homeostasis disequilibrium could have led to alterations in dopamine metabolism GSH in Huntington's disease animal models.

Dydrogesterone exerts endothelial anti-inflammatory actions decreasing expression of leukocyte adhesion molecules.

Clinical observations and basic studies show that progesterone and progestins have a variable influence on endothelial function. Dydrogesterone (DG) is a widely used progestin, but its endothelial actions have not been thoroughly assessed. In this study, we investigated the effects of DG and its metabolite 20-α-dihydro-dydrogesterone (DHD), natural progesterone as well as medroxyprogesterone acetate, on the expression of leukocyte adhesion molecules in human endothelial cells using an in vitro experimental endothelial inflammation system. Our findings show that all progestins significantly suppress endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule-1 (ICAM-1) induced by bacterial lypopolysaccharide (LPS). These inhibitory effects of DG and DHD require activation of progesterone receptor. DG and DHD decrease adhesion molecule expression associated with LPS administration by preventing nuclear translocation of the pro-inflammatory transcription factor nuclear factor-κB. In addition, DG and DHD do not alter the anti-inflammatory effects of 17ß-estradiol. In conclusion, DG and DHD decrease endothelial inflammatory responses induced by LPS, via reduced expression of the pro-atherogenic adhesion molecules VCAM-1 and ICAM-1. These actions may be relevant for the vascular effects of DG.

Biotechnological production of 20-alpha-dihydrodydrogesterone at pilot scale.

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 µM day(-1) was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.

Selectivity and potency of the retroprogesterone dydrogesterone in vitro.

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17ß-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α-dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α-dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α-dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17ß-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar K(i) values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone.

Is maternal progesterone actually independent of the fetal steroids?

Progesterone and estradiol are the foremost steroid hormones in human pregnancy. However, the origin of maternal progesterone has still not been satisfactorily explained, despite the generally accepted opinion that maternal LDL-cholesterol is a single substrate for placental synthesis of maternal progesterone. The question remains why the levels of progesterone are substantially higher in fetal as opposed to maternal blood. Hence, the role of the fetal zone of fetal adrenal (FZFA) in the synthesis of progesterone precursors was addressed. The FZFA may be directly regulated by placental CRH inducing an excessive production of sulfated 3beta-hydroxy-5-ene steroids such as sulfates of dehydroepiandrosterone (DHEAS) and pregnenolone (PregS). Due to their excellent solubility in plasma these conjugates are easily transported in excessive amounts to the placenta for further conversion to the sex hormones. While the significance of C19 3beta-hydroxy-5-ene steroid sulfates originating in FZFA for placental estrogen formation is mostly recognized, the question "Which maternal and/or fetal functions may be served by excessive production of PregS in the FZFA?" - still remains open. Our hypothesis is that, besides the necessity to synthesize de novo all the maternal progesterone from cholesterol, it may be more convenient to utilize the fetal PregS. The activities of sulfatase and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) are substantially higher than the activity of cytochrome P450scc, which is rate-limiting for the placental progesterone synthesis from LDL-cholesterol. However, as in the case of progesterone synthesis from maternal LDL-cholesterol, the relative independence of progesterone levels on FZFA activity may be a consequence of substrate saturation of enzymes converting PregS to progesterone. Some of the literature along with our current data (showing no correlation between fetal and maternal progesterone but significant partial correlations between fetal and maternal 20alpha-dihydroprogesterone (Prog20alpha) and between Prog20alpha and progesterone within the maternal blood) indicate that the localization of individual types of 17beta-hydroxysteroid dehydrogenase is responsible for a higher proportion of estrone and progesterone in the fetus, but also a higher proportion of estradiol and Prog20alpha in maternal blood. Type 2 17beta-hydroxysteroid dehydrogenase (17HSD2), which oxidizes estradiol to estrone and Prog20alpha to progesterone, is highly expressed in placental endothelial cells lining the fetal compartment. Alternatively, syncytium, which is directly in contact with maternal blood, produces high amounts of estradiol and Prog20alpha due to the effects of type 1, 5 and 7 17?-hydroxysteroid dehydrogenases (17HSD1, 17HSD5, and 17HSD7, respectively). The proposed mechanisms may serve the following functions: 1) providing substances which may influence the placental production of progesterone and synthesis of neuroprotective steroids in the fetus; and 2) creating hormonal milieu enabling control of the onset of labor.

Effects of fulvestrant alone or combined with different steroids in human breast cancer cells in vitro.

OBJECTIVES: Fulvestrant is an estrogen receptor (ER) antagonist that binds, blocks and degrades the estrogen receptor and is currently used in adjuvant treatment in postmenopausal women with ER-positive breast cancer as an alternative for tamoxifen. As an antagonist, it may induce or aggravate climacteric symptoms. In order to alleviate these symptoms, one could consider hormone therapy. The objective of this study was to analyze the effect of fulvestrant alone or in combination with different steroids in human breast cancer cells in vitro, and to demonstrate whether these steroids will compromise the efficacy of fulvestrant in ER-positive breast cancer cells. METHODS: We performed experiments in vitro with various hormone therapy preparations (estradiol (E2), dihydrodydrogesterone (DHD) and tibolone) at a concentration of 10(-6) mol/l alone or combined with fulvestrant in different breast cancer cell lines, ER-positive and ER-negative. After an incubation of 144 h, proliferation and apoptosis were measured. The first was measured by quantification of the expression of cyclin D1 mRNA, the latter by the Nicoletti fragmentation assay. RESULTS: This in vitro study revealed clear differences in results when various hormone therapy preparations, alone or combined with fulvestrant, are added to ER-positive and ER-negative breast cancer cell lines. CONCLUSIONS: Our study demonstrated that fulvestrant, an ER antagonist used in the treatment of ER-positive breast cancer, combined with E2 and DHD or in combination with tibolone, is not compromised in its efficacy in inducing apoptosis in ER-positive breast cancer cell lines in vitro.

Effects of dydrogesterone and of its stable metabolite, 20-alpha-dihydrodydrogesterone, on nitric oxide synthesis in human endothelial cells.

OBJECTIVE: To investigate the effects of P, medroxyprogesterone acetate (MPA), and dydrogesterone (DYD) and its metabolite, 20-alpha-dihydrodydrogesterone (DHD) on endothelial synthesis of nitric oxide (NO) and characterize the signaling events recruited by these compounds. The Women's Health Initiative trial reports an excess of heart disease in postmenopausal women receiving MPA. DESIGN: Cell culture. SETTING: Research laboratory. PATIENT(S): Human endothelial cells from umbilical vein. INTERVENTION(S): Treatments with P, MPA, DYD, or DHD. MAIN OUTCOME MEASURE(S): Measure of NO release, endothelial nitric oxide synthase (eNOS) activity and expression, and activation of ERK 1/2 and Akt. RESULT(S): The administration of DYD alone or in combination with estrogen to endothelial cells results in neutral effects on NO synthesis and on the activity and expression of eNOS. In parallel, the stable metabolite DHD acts similarly to natural P, enhancing the expression of eNOS and inducing rapid activation of the enzyme through the regulation of the ERK 1/2 mitogen-activated protein kinase cascade. 20-Alpha-dihydrodydrogesterone and P also potentiate eNOS induction by E2. On the contrary, MPA does not trigger eNOS enzymatic activation and decreases the extent of eNOS induction by E2. CONCLUSION(S): These findings support the concept that synthetic progestins act differently on vascular cells and that hormonal preparations may differ as to their cardiovascular effects.

Dydrogesterone (Duphaston) and its 20-dihydro-derivative as selective estrogen enzyme modulators in human breast cancer cell lines. Effect on sulfatase and on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity.

Estradiol (E2) is one of the main factors which control the growth and evolution of breast cancer. Consequently, to block the formation of E2 inside cancer cells has been an important target in recent years. Breast cancer cells possess all the enzymatic systems (e.g. sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase [17beta-HSD]) involved in the conversion of estrogen precursors into E2. Sulfotransferase, which converts estrogen to its sulfate, is also present in this tumoral tissue. Duphaston is a synthetic progestogen with properties similar to the natural progesterone. In the present study we examined the effect of Duphaston and its 20-dihydro-metabolite on the sulfatase and 17beta-HSD activities in MCF-7 and T-47D breast cancer cells. The cells were incubated with estrone sulfate (E1S) (5x10(-9)M) in the absence or presence of Duphaston or its 20-dihydro-metabolite (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. In another series of experiments, estrone (E1) (5x10(-9)M) was incubated with T-47D cells in the absence or presence of the two progestogens (5x10(-5) to 5x10(-9)M) for 24h at 37 degrees C. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. Duphaston and its 20-dihydro-metabolite, at concentrations of 5x10(-7) and 5x10(-5)M, inhibited the conversion of E1S to E2 by 14% and 63%, 65% and 74%, respectively, in MCF-7 cells; the values were 15% and 48% and 31% and 51%, respectively, in T-47D cells. In another series of experiments it was observed that, after 24-h incubation, E1 (5x10(-9)M) was converted in a great proportion to E2 in the T-47D cells and that this transformation was significantly inhibited by Duphaston and its 20-dihydro-metabolite. The IC50 value, corresponding to 50% of the inhibition in the conversion of 1 to E2, was 9x10(-6)M for 20-dihydro-metabolite in this cell line. It was concluded that the progestogen Duphaston and its 20-dihydro-metabolite are potent inhibitory agents on sulfatase and 17beta-HSD activities in breast cancer cells. Duphaston is a progestogen with properties similar to the endogenous progesterone. The data open interesting perspectives to study the biological responses of these progestogens in clinical trials of patients with breast cancer.

Comparison of the action of 17 beta-estradiol and progestins with insulin-like growth factors-I/-II and transforming growth factor-beta 1 on the growth of normal adult human bone-forming cells.

Endogenous growth factors may be involved in the prevention of bone loss by estrogen and progestins in postmenopausal women. The present study was performed to compare the action of estrogen/progestins on bone-derived cells with the effects of exogenously added purified growth factors. Human osteoblast-like (HOB) cells were incubated with 17 beta-estradiol (E2), progesterone (P), dydrogesterone (DD), 20 alpha-dihydroxydydrogesterone (DHD), with and without the growth factors, insulin-like growth factors-I/-II (IGF-I/-II) or transforming growth factor-beta type 1 (TGF-beta 1) for 24 h under serum-free conditions. Cell growth and DNA synthesis were assessed by spectophotometrical analysis of total cell number and immunochemical detection of BrdU incorporation, respectively. Compared with the sex steroids, incubation of the cells with IGF-I or TGF-beta 1 resulted in at least a two-fold increase of total HOB cell numbers. No difference in stimulating HOB growth was observed between IGF-II and the female sex steroids E2 and P. Combining IGF-I/-II or TGF-beta 1 with either E2 or P did not result in a significantly further increase in the human osteoblast-like cell growth. In conclusion, the bone anabolic growth factors, IGF-I and TGF-beta 1, may be more important regulators of osteoblast proliferation than the female sex steroids. An interaction of estrogen/gestagens with the growth factors IGF-I/-II or TGF-beta 1 was not evident from the growth of human bone-forming cells in short-term cultures.

[Contraceptive activity and mechanism of action of combinations of 6-methyl-substituted D'6-pentaranes and mestranol]. / Kontratseptivnaia aktivnost' i mekhanizm deistviia kompozitsii 6-metilzameshchennykh D'6-pentaranov i mestranola.

It has been shown in experiments on rats, mice and rabbits that 6 alpha-methyl-16 alpha, 17 alpha-cyclohexano-pregna-4-en-3, 20-dion and 6-methyl-16 alpha, 17 alpha-cyclohexano-pregna-4,6-dien-3,20-dion belonging to the series of D'6-pentaranes exhibit high contraceptive activity when combined with mestranol. The contraceptive activity of D'6-pentaranes combined with mestranol arises primarily from their inhibitory effect on luteinizing function of the hypophysis.