Circulating Hormones and Mammographic Density in Premenopausal Women.

Prior research suggests that several endogenous hormones in premenopausal women are associated with breast cancer risk; however, few studies have evaluated associations of endogenous hormones with mammographic density (MD) in premenopausal women. We conducted a cross-sectional study of plasma hormone levels in relation to MD among 634 cancer-free premenopausal women in the Nurses' Health Study II. We measured percent MD from screening mammograms using a computer-assisted method. We assayed estradiol, estrone, and estrone sulfate in blood samples timed in early follicular and mid-luteal phases of the menstrual cycle as well as testosterone, androstenedione, progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, sex hormone-binding globulin (SHBG), and anti-Müllerian hormone in luteal or untimed samples. We used multivariable linear regression to quantify the association of %MD with quartiles of each hormone, adjusting for age, body mass index, and breast cancer risk factors. Women in the highest quartile of follicular estradiol levels had significantly greater %MD compared to those in the lowest quartile [difference, 6.7 percentage points; 95% confidence interval (CI) 2.2, 11.3; p-trend < 0.001]. Similar associations were observed for follicular free estradiol but not luteal-phase estradiol. Also, women in the top (vs. bottom) quartile of free testosterone had significantly lower %MD (difference, - 4.7; 95% CI - 8.7, - 0.8; p-trend = 0.04). Higher SHBG was significantly associated with higher percent MD (difference, 4.8; 95% CI 1.1, 8.6; p-trend = 0.002). Percent MD was not strongly associated with other measured hormones. Results were similar in analyses that excluded women with anovulatory cycles. Our findings suggest that follicular estradiol and SHBG may play an important role in premenopausal percent MD.

The metabolisms of agaritine, a mushroom hydrazine in mice.

The mushroom hydrazine agaritine was measured in mouse plasma and urine using LC/MS/MS, which is highly specific. Agaritine concentration peaked 20 min after oral administration to mice (4.0 and 40 mg/kg). The concentration gradually decreased and returned to the basal level in 100 min. The maximum concentration, the time to the maximum concentration, and the half life were 0.37 microg/ml plasma, 0.33 h, and 0.71 h, respectively after administration of agaritine at 40 mg/kg body weight. One agaritine metabolite was found in the plasma and the urine from agaritine-administered mice. The structure of metabolites of agaritine by gamma-GT was next investigated using LC/MS. HMPH proved to be generated from agaritine. The oxidative stress marker 8-OHdG was detected in agaritine-administered mouse urine. After administration, the 8-OHdG level immediately tripled, and then decreased to the control level over 48 h. Its level then elevated again and remained high for 11 days. These results suggest that agaritine quickly metabolizes and disappears in the plasma, whereas DNA damage lasts for a long time after a single administration of agaritine to mice.

Fate of the mushroom hydrazine agaritine in the rat and mouse.

The fate of the mushroom hydrazine [14C]agaritine was investigated in the mouse and rat strains previously employed in carcinogenicity studies with the edible mushroom Agaricus bisporus. Agaritine was rapidly absorbed in both species, achieving higher blood levels in the mouse, but with similar area under the curve. Covalent binding of agaritine material to proteins was detected only in the liver and kidney, but the extent of binding was the same in the rat and mouse. Most of the radioactivity was excreted during the first 24 hours in both animal species: in the rat it was distributed equally between urine and feces, whereas in the mouse more of the radioactivity was excreted in the urine. No qualitative differences in the metabolic profile were evident, but quantitative differences were observed. Treatment of the urine with deconjugating enzymes did not reveal the presence of any conjugates. Agaritine, N'-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-(hydroxymethyl)benzene diazonium ion were not detected in the urine or in the plasma of either species. No mutagens or promutagens were detected by the Ames mutagenicity assay in the urine of either species after exposure to agaritine. Repeated administration of agaritine to rats and mice did not alter the urinary metabolic profile and excretion of radioactivity. Similarly, feeding mice a raw mushroom diet, according to the protocol employed in the carcinogenicity studies, did not modulate the excretion of radioactivity or the urinary metabolic pattern. No major species differences in the fate of agaritine in rat and mouse were noted that could provide a rationale for the carcinogenicity of A. bisporus in the mouse, but not in the rat.

Characteristics of peripheral blood monocytes and bone marrow macrophages from rats treated with mitomycin C, 5-fluorouracil or phenylhydrazine.

We examined the cellular functional changes in bone marrow macrophages obtained from rats treated intraperitoneally with mitomycin C (MMC), subcutaneously with 5-fluorouracil (5FU) or intraperitoneally with phenylhydrazine (PHZ) for 7 days to define the mechanisms of toxicity in the bone marrow. The functions of peripheral blood monocytes and bone marrow macrophages were also compared. MMC and PHZ caused anemia, and MMC and 5FU leukocytopenia. Changes in nucleated cell counts were different among the three drug-treatment groups, and MMC- and PHZ-treated bone marrow macrophages were activated for responsiveness to interleukin-1 (IL-1), and their migration activity, intracellular components and enzyme activities were increased. On the other hand, cellular functions of bone marrow macrophages of rats treated with 5FU were suppressed.

Decreased G3PDH binding to erythrocyte membranes in sickle cell disease.

Several membrane abnormalities have been described in red cells from patients with sickle cell disease, responsible for chronic hemolytic anemia. We describe here a 35-50% inhibition of the binding of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) to the membrane of sickle red cells. Varying the phosphorylation state of the membrane proteins did not change their affinity for the enzyme. Protein band 3 and the cytoplasmic domain of this protein isolated from sickle red cells showed normal interaction with the enzyme. The inhibition observed with intact membranes is not due to short term oxidation of membrane proteins, as various procedures inducing acute oxidative stress in normal membranes did not reproduce the inhibition of G3PDH binding. We conclude that the alteration of the binding of G3PDH to the membrane of sickle erythrocytes is probably related to long term processes involving cycles of HbS polymer formation.

In vitro effect of plasma during phenylhydrazine-induced erythrocyte oxidative damage.

Erythrocytes were incubated for 60 min either in plasma or phosphate-buffered saline containing 10 mM phenylhydrazine hydrochloride. Plasma prevented the decrease in membrane fluidity observed in saline-phenylhydrazine incubated erythrocytes but these cells showed decreases in both filterability and active extrusion of Na+ that were nearly 100% lower than those found in erythrocytes incubated in plasma-phenyl-hydrazine. Also erythrocyte lipid peroxidation, as measured as thiobarbituric acid reactive products, was 100% higher in the presence of plasma. These results suggest that plasma could play "in vivo" an active role during oxidant-induced erythrocyte damage, contributing significantly to the hemolytic effects of oxidation.

The haemolytic reactions of 1-acetyl-2-phenylhydrazine and hydrazine: a spin trapping study.

Free radical production from the reaction of hydrazine and 1-acetyl-2-phenylhydrazine (AcPhHZ) with oxyhaemoglobin and with human red blood cells, has been observed by the electron spin resonance technique of spin trapping. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the free radical intermediates detected depended on the hydrazine derivative, oxyhaemoglobin and the oxyhaem/hydrazine derivative concentration ratio. The reaction of hydrazine with oxyhaemoglobin in the presence of DMPO gave a nitroxide which was identified as a reduced dimer of DMPO. Whereas hydrazine-treated red blood cells, in the presence of DMPO, gave a nitroxide spin adduct which was identified as the hydroxyl radical spin adduct of DMPO, 5,5-dimethyl-1-pyrrolidino-1-oxyl (DMPO-OH). The reaction of AcPhHZ with oxyhaemoglobin, in the presence of DMPO, gave DMPO-OH, the phenyl radical spin adduct of DMPO, 5,5-dimethyl-2-phenylpyrrolidino-1-oxyl (DMPO-Ph) and an oxidised derivative of DMPO, 5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX). The amounts of DMPO-Ph, DMPO-OH and DMPOX observed depended on the 1-acetyl-2-phenyl-hydrazine/oxyhaemoglobin concentration ratio; DMPOX replaced DMPO-OH as the concentration of AcPhHZ was decreased. DMPOX production has been previously associated with the production of highly oxidised haem iron-oxygen intermediates. AcPhHZ treated red blood cells gave DMPO-Ph and DMPO-OH spin adducts in the presence of DMPO. DMPO had little to no effect on the rate of oxygen consumption by oxyhaemoglobin with hydrazine and AcPhHZ. Moreover, the rate of oxyhaemoglobin oxidation induced by hydrazine, was not decreased by DMPO whereas the rate of oxyhaemoglobin oxidation induced by AcPhHZ was decreased approx. 40% by DMPO. DMPO (10 mM) gave a small decrease in haemolysis and lipid peroxidation induced by 1 mM hydrazine and AcPhHZ in a 1% suspension of red blood cells.

Experiences in dealing with drug-related bound residues.

Although more than 90% of the radioactivity was excreted in the urine and feces within 10 days of oral treatment of sheep with a single dose of p [14C]toluoyl chloride phenylhydrazone (TCPH), persistent blood residues (5-6 ppm) were observed for at least 21 days. The 14C residues were largely localized in erythrocytes and covalently bound to both heme and globin. Only the phenyl group of the phenylhydrazine part of TCPH was present as 14C bound residues. An analytical procedure to measure the level of phenyl groups incorporated in heme, based on their oxidation to benzoic acid, was developed to monitor residues in treated animals. Relay metabolism in rats was studied by feeding sheep blood containing 14C residues form [14C]TCPH treatment. No retention of 14C residues in rat tissues was observed, which contrasted with the TCPH metabolism. A 90-day relay toxicity study in rats, which were fed dried blood from treated sheep containing up to 2,000 times the potential exposure to residues in the human diet, indicated no observable toxic responses. It is concluded that these data support a tolerance of 6 ppm TCPH equivalents in blood.