Perinatal exposure to xenoestrogens impairs mammary gland differentiation and modifies milk composition in Wistar rats

The current study examined the consequences of perinatal (gestation+lactation) exposure to Bisphenol A (BPA) or diethylstilbestrol (DES) on F1 mammary gland (MG) differentiation. BPA (0, 0.7 or 64 μg/kg bw/day) or DES (6 μg/kg bw/day) was administered in the drinking water of F0 rats from gestational day 9 (GD9) until weaning. F1 females were bred, MG samples obtained on GD18 and GD21, and, during lactation, milk yield and milk protein composition were assessed. On GD18, there was a decrease in α-lactalbumin and β-casein levels that was accompanied by reduced prolactin receptor and Stat5a/b expression. On GD21, delayed histological MG differentiation was observed. β-Casein levels remained decreased on GD21 and in milk samples. Moreover, the BPA- and DES-exposed groups had an altered milk yield pattern during lactation. The long-lasting effects of perinatal exposure to low doses of xenoestrogens included delayed MG differentiation, altered milk yield and modified milk composition.     

Determination of diphenhydramine in rat milk and plasma and its effects on milk composition and mammary gland nucleic acids

To study the excretion of diphenhydramine into rat milk, milk and plasma concentrations of diphenhydramine were determined in lactating rats after single or multiple oral doses. Four hours after a single dose of 40 or 100 mg/kg of diphenhydramine, milk concentrations of the drug averaged 0.30 and 2.2 micrograms/mL, respectively, in two experiments, and the milk:plasma ratios ranged from 4.4 to 7.5. Multiple doses did not significantly affect the plasma or milk concentrations or the milk:plasma ratios, which were similar to the theoretical milk:plasma ratio based on pH partitioning for this compound (i.e., 4.0). Although the concentration of diphenhydramine was higher in milk than in plasma, the estimated dose received by the pups (0.057 mg/kg/d) based on the milk concentrations was much lower than that given to the mother. Oral diphenhydramine treatment at doses which significantly reduced maternal food consumption had no effect on milk solid, lipid, protein, or lactose concentrations, nor on mammary gland RNA or DNA content, indicating that diphenhydramine did not adversely affect lactation.     

Altered mammary gland development in male rats exposed to genistein and methoxychlor.

Genistein (GE) is a prevalent phytoestrogen whose presence in human and animal foods may affect biological actions of synthetic endocrine active compounds. We have previously reported that in utero and lactational exposure to high doses of GE or the endocrine active pesticide methoxychlor (MXC) caused mammary epithelial proliferation in 21-day-old male rats. Combined exposure to GE and MXC resulted in significant feminization of the male mammary glands. The goals of the current study were to evaluate mammary responses to GE and MXC at the adult stage and investigate relevant mechanisms. Following in utero, lactational exposure (through maternal diet), and direct dietary exposure, the inguinal mammary gland of male rats (90 days of age) was found to exhibit significant morphological alterations in the groups treated with GE and/or MXC compared to the control. GE exposure (at 300 and 800 ppm concentrations) caused lobular enlargement and epithelial proliferation, whereas MXC exposure (800 ppm) led to ductal elongation and lobular enlargement. Combining the two treatments caused prominent proliferation of both ducts and alveoli; secretory material was seen in readily recognizable alveolar lumens, which are absent in untreated male mammary. We also surveyed gene expression in the mammary tissue using a cDNA microarray and evaluated relevant protein factors. The results indicated that the treatment effects are likely due to interactions between steroid hormone receptor-mediated signals and growth factor-driven cellular pathways. The distinctive responses associated with the GE+MXC combination were likely linked to enhanced actions of insulin-like growth factor 1 and related downstream pathways.     

Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland

Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 +/- 23 micrograms/ml DEHP and 25 +/- 6 micrograms/ml MEHP (mean +/- SE), while the plasma contained less than 0.5 micrograms/ml DEHP and 75 +/- 12 micrograms/ml MEHP. The high milk/plasma ratio for DEHP (greater than 200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups.     

柴胡疏肝汤对实验性乳腺增生大鼠的治疗作用

目的通过动物实验验证柴胡疏肝汤抗乳腺增生的作用。方法成熟雌性大鼠每天腹腔注射苯甲酸雌二醇0.8mg.kg-1,连续注射25d,继而连续5d腹腔注射黄体酮4mg.kg-1,造成乳腺增生模型。灌服柴胡疏肝汤提取液30d,末次给药1h,测定第二对乳头高度及直径,采血测定血清中雌激素和孕激素水平。结果柴胡疏肝汤能抑制模型大鼠的乳头高度及直径,降低造模大鼠血清中雌激素水平。结论柴胡疏肝汤对大鼠实验性乳腺增生具有明显的治疗作用。     

Gene expression profiles in mammary gland of male rats treated with genistein and methoxychlor

Humans and wildlife are frequently exposed to mixtures of natural and synthetic endocrine-active compounds. To understand the impact of dietary phytoestrogen on the susceptibility to synthetic chemicals in the environment, we studied the effects of a binary mixture consisting of the isoflavone genistein and the pesticide methoxychlor on the development of the mammary gland. Sprague-Dawley rats were exposed to genistein at 800ppm, methoxychlor at 800ppm, or their combination through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. At post-natal day (PND) 90, offspring rats were killed and their inguinal mammary glands collected for gene expression analysis utilizing the Clontech Atlas Rat 1.2 cDNA array, which contains probes for 1176 genes. Treatment with both genistein and methoxychlor altered gene expression profiles of the mammary glands in male rats, and the effects were more prominent in the combination treatment than the single-compound groups. Specific gene changes suggested that treatments affected the stromal and epithelial compartments of the mammary, involving genes controlling growth factor signaling, apoptosis, and tissue remodeling. This study demonstrates that dietary phytoestrogens in combination with a synthetic endocrine-active chemical can cause unique effects in endocrine-responsive tissues and highlights the importance of studying the effects of chemical combinations on the multiple biological processes underlying toxicological responses.     

柴青消癖胶囊对乳腺增生模型大鼠的治疗作用研究

目的探讨柴青消癖胶囊对乳腺增生模型大鼠的治疗作用及可能的作用机制。方法清洁级SD大鼠70只,随机分成7组：对照组,模型组,枸橼酸他莫昔芬组,消乳散结胶囊组,柴青消癖胶囊低、中、高剂量（0.432、0.856、1.712g/kg）组。5周后,测定乳头直径及高度、脏器指数、血清中孕激素（P）、雌激素（E2）、泌乳素（PRL）、睾酮（T）水平及超氧歧化酶（SOD）、丙二醛（MDA）量,镜下观察乳腺组织病理形态学变化。结果柴青消癖胶囊中、高剂量组能显著减小大鼠乳头直径及高度;升高胸腺、脾指数,降低子宫、卵巢指数;能提高血清中P、E2、PRL水平,降低T的量;可明显增加血清中SOD活性,降低MDA水平。结论柴青消癖胶囊对乳腺增生模型大鼠有显著的治疗作用,其作用机制可能与增强免疫力、调节血清中性激素水平及抗氧化作用有关。     

Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.     

Altered mammary gland differentiation and progesterone receptor expression in rats fed soy and whey proteins.

There are suspected links between an animal's diet, differentiation status of a target tissue, and sensitivity to chemically induced cancer. We have demonstrated that rats fed AIN93G diets made with soy protein isolate (SPI) or whey protein hydrolysate (WPH) had a lower incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced adenocarcinoma than rats fed the same diet made with casein (CAS). The current study was conducted to determine the differentiation status of the mammary glands during development. Offspring of rats (n = 5-10/group) were fed diets made with SPI, WPH, or CAS throughout life (beginning on gestation day 4) and were sacrificed on postnatal day (PND) 21, PND 33, PND 50 or on metaestrous between PND 48 and PND 51. There were no significant differences between the numbers of mammary terminal end buds (TEBs) or lobuloalveoli (LOB) between any of the diets groups at PND 21 or PND 33, but at PND 50 there was an 75% decrease in the mean numbers of TEBs/mm(2) in the SPI- or WPH-fed rats, compared with the CAS-fed rats (p = 0.09 and p = 0.06, respectively). In rats sacrificed in metaestrous, there were no significant differences in the proliferation index (PI) in the TEBs or LOB between any of the diet groups. In metaestrous rats, there were twice as many cells expressing estrogen receptor beta (ERbeta; approximately 60%) compared with estrogen receptor alpha (ERalpha; approximately 30%) in the LOB and 1.5 times more ERbeta (approximately 60%) compared with estrogen receptor alpha (ERalpha, approximately 40%) in the TEBs. There were no diet-dependent differences in expression of ERalpha and ERbeta. Similarly, there were no differences between the diet groups in progesterone receptor (PR) expressing LOB cells. However, in the TEBs there was a diet-dependent difference in PR positive cells with a 34% increase (p < 0.05) in the SPI-fed rats and a 38% increase (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. These results show that the type of dietary protein alters the phenotype of mammary epithelia in the TEBs. The SPI- and WPH-dependent changes in mammary differentiation may contribute to the reduced sensitivity to DMBA-induced mammary cancer in rats fed these proteins.     

Distribution of lead in lactating mice and suckling offspring with special emphasis on the mammary gland

 The distribution of lead in lactating mice and suckling offspring was studied with whole body autoradiography at 4 and 24 h after a single intravenous injection of ²⁰³Pb (50 nmol Pb/kg) to the dams. In the lactating mice on day 14 of lactation, the highest uptake of radioactivity at 4 h after administration was recorded in renal cortex, skeleton and liver. A high uptake was also evident in the mammary gland. At 24 h after administration, the radioactivity had decreased in most organs except in the skeleton. In the suckling pups, exposed to lead only via dams’ milk for 24 h, the highest level of radioactivity was present in the intestinal mucosa and a much lower level of radioactivity was present in the skeleton. The mammary glands from mice given three daily intravenous injections of 240 μmol Pb/kg were examined with X-ray microanalysis. At 4 h after the last injection, lead was found associated with casein micelles both inside the alveolar cell and in the milk lumen, indicating that lead is excreted into the milk, bound to casein, via the Golgi secretory system. Received 8 May 1995 / Accepted 16 July 1995     

积雪草总苷抗实验性大鼠乳腺增生

目的 :观察积雪草总苷抗实验性大鼠乳腺增生症的作用。方法 :SD大鼠 70只 ,随机分为 7组(n =10 ) :正常组、模型组、乳癖消组、他莫昔芬组、积雪草总苷 1.5 ,3,6mg·kg- 13个剂量组。采用雌二醇 (E2 )腿部肌内注射 0 .0 2 5mg·kg- 1·d- 1,连续2 5d ,改肌内注射黄体酮 5mg·kg- 1·d- 1,连续 5d ,刺激大鼠乳腺组织 ,制备乳腺增生模型。于造模同时给予积雪草总苷胃饲 ,共 30d ,观察大鼠乳头增生大小并检测E2 和黄体醇 (P)水平及大鼠乳腺组织病理形态学变化。结果 :积雪草总苷可抑制实验性大鼠乳腺组织的增生 ,积雪草总苷 3,6mg·k- 1剂量组与模型组比较均可降低血清雌激素水平 ,P <0 .0 5 ,P <0 .0 1。组织病理形态学观察 ,积雪草总苷组与模型组比较 ,腺上皮增生明显受抑制。结论 :积雪草总苷具有良好的抗实验性大鼠乳腺增生症的作用。     

Excretion of high concentrations of cimetidine and ranitidine into rat milk and their effects on milk composition and mammary gland nucleic acid content.

The excretion of cimetidine and ranitidine into rat milk following single or multiple oral doses and the subsequent effects on their suckling pups and on milk composition and milk synthesis were investigated. Following a single dose of [3H]cimetidine, peak milk cimetidine concentrations were maintained from 1 until 4 hr, while plasma concentrations peaked at 10% of the milk level at 30 min and then declined. Multiple doses of cimetidine (18 or 180 mg/kg/day) on Days 13-16 of lactation led to milk cimetidine concentrations of 17 and 113 micrograms/ml. The milk/plasma ratios far exceeded the theoretical milk/plasma ratio of 2.0. Ranitidine concentrations in rat milk following ranitidine treatment (4.5 or 45 mg/kg/day) were also greater (6.8-15 times) than in plasma, but only slightly greater than the predicted ratio of 5.0. There were no changes in liver weight or in hepatic aminopyrine N-demethylase activity in the cimetidine- or ranitidine-treated dams or their pups. Cimetidine treatment had no effect on milk lipid, solid, or protein content, but at 180 mg/kg/day, caused a significant increase in milk lactose. The RNA/DNA ratio in the mammary gland was significantly increased by cimetidine, suggesting increased milk synthesis. Ranitidine had no effect on milk composition or on mammary gland RNA, DNA, or RNA/DNA. Therefore, high concentrations of cimetidine and ranitidine were excreted into rat milk, but no deleterious effects on the suckling pups, or the composition of the milk, or on the milk synthetic activity were observed.     

Histamine in C3H/W mice carrying spontaneous tumors of the mammary gland

Adenocarcinoma mammae, a spontaneously growing mammary cancer in C3H/W mice, contrary to many transplanted tumors does not evoke any rise in histamine level either in the tumor or in distant tissues. On the other hand, the histamine level is reduced by 90% in the tumor in comparison with the healthy gland. This seems to be a consequence of the fall of histidine decarboxylase activity to below a detectable level. There is also a significant reduction in histamine N-methyltransferase activity to one-fifth of the control level. The healthy mammary gland contains a high concentration of histamine and catabolizes it exclusively through the methylation pathway.     

Mixtures of environmentally relevant endocrine disrupting chemicals affect mammary gland development in female and male rats

Estrogenic chemicals are able to alter mammary gland development in female rodents, but little is known on the effects of anti-androgens and mixtures of endocrine disrupting chemicals (EDCs) with dissimilar modes of action.Pregnant rat dams were exposed during gestation and lactation to mixtures of environmentally relevant EDCs with estrogenic, anti-androgenic or dissimilar modes of action (TotalMix) of 100-, 200- or 450-fold high end human intake estimates. Mammary glands of prepubertal and adult female and male offspring were examined.Oestrogens increased mammary outgrowth in prepubertal females and the mRNA level of matrix metalloproteinase-3, which may be a potential biomarker for increased outgrowth. Mixtures of EDCs gave rise to ductal hyperplasia in adult males. Adult female mammary glands of the TotalMix group showed morphological changes possibly reflecting increased prolactin levels. In conclusion both estrogenic and anti-androgenic chemicals given during foetal life and lactation affected mammary glands in the offspring.     

Effects of genistein on the mammary gland proliferation of adult ovariectomised Wistar rats

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E(2), does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.     

Cadmium exposure and trace elements in human breast milk

The interrelations of the seven elements, calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), phosphorus (P), copper (Cu), zinc (Zn), and cadmium (Cd) in human breast milk were examined in Japanese mothers to clarify the effects of Cd exposure on these important elements for infant growth. Breast milk and urine samples were obtained from 68 mothers, aged 19-38 years, at 5-8 days postpartum. The concentrations were determined by inductively-coupled plasma atomic emission spectrometry for Ca, Mg, Na, K, P, by flame atomic absorption spectrophotometry for Cu and Zn, and by flameless atomic absorption spectrophotometry for Cd. Geometrical mean Cd concentrations were 0.28 (geometrical standard deviation=1.82) microg/l in breast milk and 1.00 (1.93) microg/g creatinine in urine. Among the above elements only Cd concentration in breast milk was significantly correlated with urinary Cd concentration (r=0.451, P<0.001). Significant positive correlations were found between Cu and Ca (r=0.500, P<0.001), Cu and Mg (r=0.378, P<0.01), and Zn and Mg (r=0.355, P<0.01) in breast milk. Cd concentration in breast milk showed an inverse relationship with Ca concentration in breast milk (r=-0.248, P<0.05). These results indicate that the Cd concentration in breast milk closely reflects Cd body burden, with increased Cd in breast milk possibly affecting Ca secretion in breast milk.     

Cadmium concentrations in milk and blood of smoking mothers

Cadmium concentrations were measured by flameless atomic absorption spectrometry in blood and mature milk of 15 nonsmoking and 56 smoking mothers during the nursing period. Both blood and milk concentrations increased with increasing cigarette consumption. The median blood and milk concentrations in nonsmokers were 0.54 and 0.07 microgram/l, respectively; these values rose to 1.54 and 0.16 micrograms/l in blood and milk of mothers smoking more than 20 cigarettes per day. Milk concentrations of cadmium were approximately 10% of corresponding blood concentrations. The cadmium exposure of infants nursed by nonsmoking as well as by smoking mothers was far below the exposure of formula-fed infants or the provisional acceptable weekly intake level set by the WHO.