Urinary phytoestrogens and postmenopausal breast cancer risk.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estradiol. We report the associations of two major phytoestrogens, genistein and enterolactone, with breast cancer risk, using urinary specimens collected 1-9 years before breast cancer was diagnosed. The subjects were 88 breast cancer cases and 268 controls, selected from a cohort of postmenopausal women (n = 14,697) who participated in a breast cancer screening program. Mean levels of urinary genistein and enterolactone were determined by time resolved fluoroimmunoassay, using an average of two overnight urinary samples obtained from each participant on the first and the second screening rounds with a time interval of approximately 1 year. Odds ratios (ORs) of the highest to the lowest tertile of urinary phytoestrogen/creatinine concentrations and 95% confidence intervals (CIs) were computed. Higher urinary genistein excretion was weakly and nonsignificantly associated with a reduced breast cancer risk. OR for the highest tertile compared with lowest tertile was 0.83; 95% CI, 0.46-1.51. Higher urinary enterolactone excretion was weakly and nonsignificantly associated with an increased breast cancer risk. OR for the highest tertile compared with the lowest tertile was 1.43; 95% CI, 0.79-2.59. Tests for trends for both phytoestrogens were nonsignificant. We were not able to detect the previously reported protective effects of genistein and enterolactone on breast cancer risk in our postmenopausal population of Dutch women. Such an effect may be smaller than expected and/or limited to specific subgroups of the population.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist.

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists). This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples. The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELSA results. Four calves were orally treated with RCT (0.1 mg kg-1 body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli). High concentrations of RCT residues were found throughout the medication period (44-473 ng ml-1; LC-MS-MS data) and remained present for several days following removal of the drug from the diet. RCT residues were no longer detectable 2 weeks after withdrawal. Good agreement (r2 = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for sets of analyses. The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal. However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: a possible way forward.

The drug salbutamol (SBL) is a beta-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.

Effects of wheat bran on blood and tissue hormone levels in adult female rats.

The protective effect of dietary fiber on breast cancer development might be explained by the interaction between dietary fiber and hormonal processes. We studied the effects of dietary fiber and the effects of a reduced energy intake on the exposure of mammary tissue to both estrogens and progesterone, as well as the blood plasma levels of these steroids and of LH and FSH. Adult female Fisher rats were fed ad libitum either a low-fiber diet (0.5% dietary fiber based on wheat flour) or a high-fiber diet (9.2% dietary fiber based on wheat bran). A third group was used to control for the reduced energy intake of the high-fiber group and was fed the low-fiber diet restricted. Energy intake was similar for the second and third groups. Four out of 14 rats of the high-fiber group and 4 out of 15 rats of the restricted low-fiber groups were not in cycle after seven weeks on the experimental diets, indicating that the estrous cycle was significantly affected by a reduced energy intake. Exposure of mammary tissue to estrogens did not differ among the groups, as measured by estrone, estradiol-17 beta, estriol and peroxidase activity. During the peak period, plasma LH levels were significantly higher in the high-fiber group than in the two low-fiber groups. FSH and progesterone plasma levels were unaffected by the experimental diets. It is concluded that dietary fiber affects the hormonal processes involved in breast cancer development. The increased LH levels indicate an increased estrogen production in the ad libitum high-fiber group.

Effects of wheat bran and energy restriction on onset of puberty, cell proliferation and development of mammary tissue in female rats.

Delayed onset of puberty and mammary development is assumed to reduce the risk of mammary cancer. An animal experiment was performed to investigate the influence of dietary fiber, which is known to affect hormonal balance, on these characteristics. Forty-five immature female rats were randomized into three groups, which were fed ad libitum a low-fiber diet (less than 0.5% dietary fiber based on white wheat flour), a high-fiber diet (9.2% dietary fiber based on wheat bran), or an energy-restricted low-fiber diet providing 90% of the energy of the ad libitum low-fiber diet. Energy intake in the second and third groups was similar. Wheat bran slightly delayed onset of puberty, whereas restricted energy intake delayed onset of puberty by about six days. At 48-58 days of age, 14 rats of the low-fiber group, 10 of the high-fiber group and 7 of the restricted group were in cycle. Development of mammary tissue was rudimentary in rats of the energy-restricted low-fiber group, stronger in the high-fiber group and strongest in the ad libitum low-fiber group. Cell proliferation in mammary tissue was similar for both groups fed ad libitum, but significantly lower in the restricted group. Peroxidase activity, a biomarker for estrogenicity, was lower in the high-fiber group than in the two low-fiber groups. It is concluded that wheat bran and, even more effectively, an imposed restricted energy intake delays onset of puberty and mammary development. This shortens the time for mammary cells to be initiated to tumor cells and hence reduces the risk of mammary cancer development.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of wheat bran on excretion of radioactively labeled estradiol-17 beta and estrone-glucuronide injected intravenously in male rats.

Urinary and fecal estrogen excretion were studied in male rats fed a non-fiber wheat starch diet (dietary fiber less than 1%; NF group; n = 4), a low-fiber wheat flour diet (dietary fiber 2%; LF group; n = 4) or a high-fiber wheat bran diet (dietary fiber 11.6%; HF group; n = 3). Short-term effects of the experimental diet on estrogen excretion were studied after i.v. injection of 5 microCi (0.185 MBq) of [14C]estradiol-17 beta (E2) into the tail vein of the rats fed the diets for 2 days. After 3 weeks on the experimental diets, the long-term effects were studied after injection of 5 microCi of [14C]E2 and 10 microCi of [3H]estrone-3-glucuronide (E1-gluc). The diet was found to affect estrogen excretion. The short-term effect indicated that rats fed the HF diet excreted a relatively large amount of labeled compounds in the feces during the first day after injection, while rats fed the NF or the LF diets excreted about half that amount over the same period. On the other hand, urinary excretion of labeled compounds was significantly higher in the NF and LF rats. The long-term effect resulted in steeper slopes (P less than 0.05) of the fecal excretion profiles of rats fed the HF diet as compared with rats fed the NF and LF diets, indicating an accelerated fecal excretion of labeled compounds in the HF rats. The kinetic profiles of 14C and 3H radioactivity in blood plasma indicated a fast decrease (t1/2 of less than 2 min) for both [14C]E2 and [3H]E1-gluc. It was concluded that, owing to the short-term effect of wheat bran intake, during the first 24 h after i.v. administration relatively large amounts of radioactively labeled compounds are excreted in feces of rats fed the HF diet. In contrast, excretion is lower in urine of these rats. When the microflora is adapted to the experimental diet the wheat bran diet still results in an accelerated fecal excretion of labeled compounds, which might be attributed to an interruption of the enterohepatic circulation of estrogens. This might result in lowered plasma and/or tissue estrogen levels and hence a decreased exposure of estrogen-sensitive tissue to estrogens, which might decrease risk on mammary (breast) cancer development.

Influence of wheat bran on NMU-induced mammary tumor development, plasma estrogen levels and estrogen excretion in female rats.

In our animal experiments the hypothesis was tested that a high-fiber (HF) diet reduces tumor promotion by interruption of the enterohepatic circulation resulting in lowered estrogen exposure of the estrogen-sensitive tissue. In the first experiment the development of N-nitrosomethylurea (NMU) induced mammary tumors was investigated. One group of rats (HF) was fed a HF diet (11% fiber, based on wheat bran), the other group (LF) fed a low-fiber diet (0.5% fiber, based on white wheat flour). Tumor incidence (90 and 80%, respectively) and latency (121 and 128 days, respectively) were similar in the HF and LF groups. Compared to the LF group, HF rats had lower tumor weights (0.16 vs 0.55 g; P less than 0.01) and a slightly lower tumor multiplicity (1.8 vs 2.8 tumors per tumor-bearing rat). These differences were reduced after adjustment for body weight. In a second experiment rats, not treated with the carcinogen, were kept on the same HF and LF diets. From these rats 24-h urine and feces and orbital blood samples were collected for analysis of (un)conjugated estrogens. The excretion of both free and conjugated estrogens in fecal samples was about 3-fold higher in HF rats than in LF rats. During the basal period of the cycle urinary excretion of estrone was lower in HF rats (mean 9.7 ng/day) than in LF rats (mean 13.0 ng/day; P less than 0.05). It is concluded that wheat bran interrupts the enterohepatic circulation of estrogens, but plasma levels are not affected. Whether the development of mammary tumors is reduced by the introduction of specific components of wheat bran, or by a reduced body weight due to a lower (effective) energy intake remains to be determined.

In vitro binding of estrogens by dietary fiber and the in vivo apparent digestibility tested in pigs.

Within the framework of experiments related to the association between dietary fiber and breast cancer an in vitro test system was used to study the binding of estrogens to various fibers (e.g. cholestyramin, lignin and cellulose) and fiber sources (e.g. wheat bran, cereals, seeds and legumes). Furthermore, the in vivo apparent digestibility of the different fiber sources was tested using a mobile nylon bag technique in intestine-cannulated pigs. Estradiol-17 beta (E2) bound more strongly to the various fibers than did estrone (E1), estriol or estrone-3-glucuronide. At increasing pH (greater than 7) binding of both E1 and E2 to wheat bran decreased significantly. Cholestyramine and lignin bound almost all estrogens present in the medium. Linseed (91%), oats (83%), barley chaff (88%) and wheat bran (82%) are other excellent binders of E2. Corn, rye and white wheat flour showed lower binding capacity with a relatively low affinity. Cereals with the highest percentage of lignin in the fiber (greater than 3%) were also the fiber sources with the lowest apparent digestibility. Estrogens bound with the highest affinity (relative to bovine serum albumin) to these fiber sources. Together with wheat bran and lignin, oats, linseed and soybean seem to be products with good perspectives for in vivo evaluation of the lowering effect of dietary fiber on estrogen exposure of estrogen-sensitive tissues.

Control system for detection of the illegal use of naturally occurring steroids in calves.

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17 beta (E2-17 beta), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic-radioimmunoassay method, reference values were established for concentrations of E2-17 beta, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E2-17 beta levels of both male and female calves were less than 0.01 microgram/l in blood plasma and less than 0.2 microgram/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were less than 0.1-0.3 micrograms/l in blood plasma and less than 0.6-10 micrograms/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E2-17 beta benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E2-17 beta and E2-17 alpha in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.

A new radioimmunoassay for the determination of the angiotensin-converting enzyme inhibitor perindopril and its active metabolite in plasma and urine: advantages of a lysine derivative as immunogen to improve the assay specificity.

A new radioimmunoassay (RIA) was developed for the direct measurement of perindoprilate (PT), the active metabolite (diacid) of Perindopril (P), an angiotensin-converting enzyme (ACE) inhibitor. Antibodies were raised in rabbits against the lysine derivative of PT conjugated to bovine serum albumin. The p-hydroxyphenyl derivative of the lysine analogue was used for preparation of the radioligand by iodination (125I). Cross-reactivities for the glucuronide metabolites of P and PT are low (0.25 and 3.5%, respectively). The theoretical limit of detection is 0.2 nM, the sensitivity attainable with random samples is about 0.5 nM. Within- and between-assay variabilities observed were 4.2-6.7 and 2.8-5.9%, respectively (concentration range 2.1-41.7 nM). Serial dilution of plasma and urine samples showed excellent parallelism (r greater than 0.95; P less than 0.001). Recoveries of PT spiked to urine and plasma samples were 90-120%. The prodrug P can be measured in the same sample (plasma/urine) after chromatographic separation on a Dowex AG 1 x 2 anion-exchange column and quantitative alkaline hydrolysis of the P-containing fraction. It is concluded that the specificity and sensitivity of this assay are amply sufficient for pharmacokinetic studies and in patient monitoring.

In vitro synthesis of 16 alpha-hydroxyestrone by female rat liver microsomes: its possible role in the etiology of breast cancer.

Liver homogenates from female rat strains (Sprague-Dawley, Wistar and Fisher) were incubated in a NADPH regenerating medium in the presence of labelled and unlabelled estrone. Liver microsomes isolated from male rats and female mice were used as positive controls. Using HPLC and paper chromatography, under the experimental conditions used it was found that liver homogenates from female rats were able to convert estrone to various metabolites such as 16 alpha-hydroxyestrone. In a mutagenicity assay (Ames test), with 16 alpha-hydroxyesterone as test substance, two strains (TA98 and TA1538) of the five strains tested showed a 2-3-fold increase in the number of his+ revertants relative to the control values. Estrone did not cause any mutagens in the test used. It is concluded that female rats are able to synthesize 16 alpha-hydroxyestron in vitro. Whether this compound is risk factor for breast cancer remains unclear.

Multi-residue screening of bovine urine on xenobiotic oestrogens with an oestrogen radioreceptor assay.

An improved radioreceptor assay (RRA) is used for the screening of urine samples from cattle for the presence of exogenous oestrogenic anabolic compounds, e.g., stilbenes and zeranol. The method includes extraction of the hormones from urine samples with simultaneous purification using reversed-phase C18 cartridges. High-performance liquid chromatography is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently analysed using the RRA with the oestrogen receptor, isolated from immature calf uteri, as a binder and tritiated 17 beta-oestradiol as a tracer. Urine samples from untreated calves and cows and samples from calves treated with zeranol-trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol were analysed with this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng/ml 'apparent' oestradiol in urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.

Oestrogen radioreceptor assay for multi-residue screening of bovine urine for oestrogenic anabolic compounds.

An improved radioreceptor assay (RRA), based on the method originally described by Korenman (1968), was used for the screening of urine samples of cattle for the presence of exogenous oestrogenic anabolic compounds, i.e. stilbenes and zeranol. The method includes extraction of the hormones from urine samples with a simultaneous purification using reversed-phase C18 cartridges. HPLC is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently quantified using the RRA with the oestrogen receptor, isolated from immature calf uteri, as binder and tritiated 17 beta-oestradiol as tracer. Urine samples from untreated calves and cows, as well as samples from calves treated with zeranol/trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol, were analysed using this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng 'apparent' oestradiol per ml urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.

Characterization of alpha-MSH-like immunoreactivity in human plasma.

We report new information on the presence of alpha-melanotropin(alpha-MSH)-like immunoreactivity in the peripheral circulation of the adult human. Pooled blood from 20 patients with non-endocrine diseases was subjected to a Sep-pak pre-purification followed by a high pressure liquid chromatographic (HPLC) fractionation. The eluate from the HPLC column was analyzed by a radioimmunoassay (RIA) specific for the C-terminal part of the alpha-MSH molecule. From this it appeared that alpha-MSH was the major alpha-MSH-like immunoreactive peptide present in human blood with a level of 2-5 pg/ml. This level is similar to the one determined by direct measurements in Sep-pak pre-purified human plasma (median 2.0 pg/ml, n = 11). Des-acetyl alpha-MSH was present in human blood in only a minor quantity. We discuss this finding in view of earlier reports on alpha-MSH-like immunoreactivity in human pituitary tissue.

A direct radioimmunoassay for the determination of adrenocorticotropic hormone (ACTH) and a clinical evaluation.

A direct radioimmunoassay for the determination of ACTH in a small volume of serum was developed using a commercially available antiserum and tracer. A single-stage radioimmunoassay with an incubation period of 4 days gave reliable and reproducible ACTH values. In the incubation medium 0.1% 2-mercaptoethanol was used as the antioxidant. During a 15-month period the assay has been used routinely in the laboratory. The coefficient of variation was less than 10% in the physiological and pathological range. A normal range of 12 to 60 ng/L was found. The reliability of the assay is demonstrated by results obtained in patients with disorders of the pituitary-adrenal axis.

Successful treatment with sodium valproate of a patient with Cushing's disease and gross enlargement of the pituitary.

Transfrontal hypophysectomy was performed in a patient with Cushing's disease and gross enlargement of the pituitary. Despite some reduction of cortisol production active Cushing's syndrome remained due to the presence of a tumour remnant. Medical treatment with the GABA-transaminase inhibitor sodium valproate induced hypocorticism necessitating corticosteroid substitution therapy. Nine months after sodium valproate withdrawal hypercorticism was documented. Re-institution of sodium valproate treatment induced hypocorticism again. As sodium valproate is known to induce a decrease of plasma ACTH in Nelson's syndrome, it is proposed that large tumours present at the time of diagnosis and those appearing after adrenalectomy may represent the spectrum of a single disorder. A prospective trial to study the effects of sodium valproate and other neurotransmitter modulating agents on the size and endocrine function of ACTH secreting macroadenomas is urgently needed.

Gel-chromatographic analysis of 99mTc-labeled human serum albumin prepared with Sn(II) as the reductant.

The labeling of human serum albumin (HSA) with 99mTc using Sn(II) as the reductant has been reinvestigated in view of the possibility of formation of Sn-Tc colloids. Preparations of 99mTc-HSA and mixtures of 99mTc-TcO-4 with Sn(II)Cl2 have been analyzed by means of gel chromatography using various gel matrices and eluents. No Sn-Tc colloids could be eluded from samples of 99mTc-HSA preparations. The percentage of labeling was about 90%. A comparative investigation of a number of gel-chromatographic systems for the detailed analysis of 99mTc-HSA has been made. It is found that a long column with Sephadex G-200 gives the best results.