Magnetic resonance imaging - evaluation of mesorectal lymphadenopathy in patients with rectal cancer.

INTRODUCTION: Multidisciplinary management of patients with rectal cancer presents a gold standard of care; neoadjuvant therapy indications are based on magnetic resonance imaging (MRI) description of the local stage of the carcinoma. Although the accuracy of MRI-based assessment of cancer depth of invasion is satisfactory, its accuracy in the assessment of mesorectal lymphadenopathy is very questionable. METHODS: This was a prospective, single-centre, cohort study focused on the accuracy of preoperative MRI in the assessment of mesorectal lymph nodes (LN). MRI findings of each patient were compared with detailed histopathological examination of rectal specimens. RESULTS: Forty patients with rectal cancer, undergoing rectal resection with total mesorectal excision were enrolled in the study. MRI assessment of the T-stage was correct in 22 of the 40 study patients (55.0%). T-stage overstaging was noted in 14 (35.0%), and understaging in 4 (10.0%) study patients. According to preoperative MRI (using Horvat's criteria), there were 50 suspicious/malignant lymph nodes. Only 13 of these 50 LNs (26.0%) were proved malignant on histopathology examination. In total, our study group included 18 patients with suspicious/positive LNs (according to preoperative MRI) who were classified as cN+. MRI diagnosis of malignant lymphadenopathy was correct in only 33.3% of these patients. CONCLUSION: MRI shows very low accuracy in the evaluation of mesorectal lymph nodes in patients with rectal cancer. Therefore neoadjuvant therapy should be offered particularly with respect to MRI description of the depth of carcinoma invasion (T-stage and relationship to fascia propria of the rectum).

Prognostic significance of miR-23b in combination with P-gp, MRP and LRP/MVP expression in non-small cell lung cancer.

Recently, miR-23b has emerged as a promising new cancer biomarker but its role in lung cancer has not been established yet. Patients still do not respond well to available treatments, probably due to expression of multidrug resistance (MDR) proteins, such as P-gp, MRP and LRP/MVP. The aim of this study was to determine the role of miR-23b in non-small cell lung cancer (NSCLC) and its relationship to the patient outcome together with MDR transporter proteins. We immunohistochemically evaluated expression of P-gp, MRP and LRP/MVP and quantified the relative levels of miR-23b in 62 NSCLC patients´ samples. The prognostic significance of miR-23b and MDR proteins was tested by Kaplan-Meier and Cox-regression analysis. Our results showed that miR-23b is mostly downregulated in NSCLC samples (57/62) and that its upregulation in tumors is connected with longer progression-free survival (PFS; P = 0.065) and overall survival (OS; P = 0.048). The Cox proportional hazard model revealed that the risk of death or relapse in NSCLC patients with miR-23b downregulation increases together with LRP/MVP expression and both risks decrease with miR-23b upregulation (HRPFS = 4.342, PPFS = 0.022; HROS = 4.408, POS = 0.015). Our findings indicate that miR-23b, especially in combination with LRP/MVP expression, might serve as a suitable prognostic biomarker for NSCLC patients.

Overexpression of filamin-A protein is associated with aggressive phenotype and poor survival outcomes in NSCLC patients treated with platinum-based combination chemotherapy.

An actin-binding protein filamin A connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Recently, it has been reported that filamin A is required for efficient regulation of early stages of DNA repair process. Moreover, some in vitro studies showed that the overexpression of filamin A determines resistance to various cytotoxic drugs, including cisplatin. We aimed to analyse the expression of filamin A protein in resected NSCLC (Non Small Cell Lung Cancer) specimens, to investigate the association of the level of filamin A protein expression and other clinicopathological features, and possible relationship between the expression of filamin A and survival outcome in NSCLC patients, treated with platinum-based combination chemotherapy. We performed filamin A protein immunohistochemistry on formalin-fixed and paraffin-embedded (FFPE) tissue sections from 135 NSCLC patients, using EP2405Y antibody against C-terminus of filamin A. Cytoplasmic, membranous and nuclear positivity of filamin A was evaluated semi-quantitatively and correlated with available clinicopathological data. Patients were divided into two groups for survival analysis (I group - patients treated with adjuvant platinum-based chemotherapy, II group - patients with surgical treatment only). We found significant positive correlation between filamin A protein expression and NSCLC stage (r=0.249; p<0,05), presence of lymph node (N)(r=0.205; p<0,05) and distant metastases (M) (r=0.332; P<0.01). Increased filamin A protein expression was significantly related with poor survival outcomes in patients with adjuvant platinum-based chemotherapy: OS (HR=1.005, 95%CI[1.000;1.010], p=0.037), DFS (HR=1.004, 95%CI [1.001:1.008], p=0,017). Multivariate Cox proportional hazards regression analysis also showed that overexpression of filamin A represents an independent risk factor for disease relapse, in addition to tumor size, stage, and metastases status (HR=1.723, 95%CI [1.021:2.909], p<0.05). Thus, filamin A expression might be a new prognostic marker in patients with NSCLC.

Ewing's sarcoma of the urinary bladder - the urologic and pathologic differential diagnosis and current therapeutic options.

BACKGROUND: Bladder cancer is 11th most common cancer worldwide. Histologically, most of the tumors are classified as urothelial carcinomas. Less common variants (squamous cell or adenocarcinomas) usually comprise up to 10% of cases. Other types of tumors are exceptional. The finding of Ewing's sarcoma in the bladder is considered extremely rare. CASE: We present the case of a 54-year-old female patient examined for painless hematuria. During the follow-up examination, a bulky tumor of the bladder was detected, but considering the extent of the bladder tumor, only a diagnostic transurethral resection was possible. According to the primary staging, the disease was already advanced at the time of admission with metastatic spread, anemia and present obstruction of the upper urinary tract. RESULTS: Histologically, Ewing's sarcoma was surprisingly demonstrated in the urinary bladder. Anemia caused by hematuria and advanced disease was corrected by blood transfusions and obstruction of the right kidney by puncture nephrostomy. However, despite a very quick diagnosis, completion of staging and preparation of the patient for further treatment, the patient had died before the planned systemic treatment began. CONCLUSION: The diagnosis of Ewing's sarcoma is identical to that of the other bladder tumors, i.e. transurethral resection. In the case of confirmation of this histological type, it is necessary to complete staging examinations and start multimodal treatment. Early systemic chemotherapy plays a key role and if metastatic spread is excluded, radical cystectomy or radiotherapy are included, too. The aim of our communication is to present a rare case of this disease, discuss the differential diagnosis and point out the principles and possibilities of its treatment.

Differentiation pathways in carcinogenesis and in chemo- and radioresistance.

Cancer stem cells (CSCs) share many features with embryonic stem cells (ESCs) such as the ability for self-renewal and differentiation. Signaling pathways that are involved in these processes are also involved in chemo- and radioresistance (e.g. Wnt, Notch and Hedgehog pathways). This review is focused on the influence of three important differentiation pathways on carcinogenesis and on chemo- and radioresistance in ESCs and CSCs.

[Biomarkers in the detection of minimal systemic dissemination in lung cancer patients]. / Biomarkery detekce minimální systémové diseminace u nemocných s karcinomem plic.

INTRODUCTION: Minimal systemic disease (MSD) means the presence of circulating or disseminated tumour cells in mesenchymal compartments of a patientts' body (lymphatic nodes, blood or bone marrow). The aim of our pilot study was to identify sensitive and specific markers for MSD detection in 50 lung cancer patients, who underwent curative surgery in the I. Department of Surgery, Faculty of Medicine and Dentistry, Palacky University and Faculty Hospital Olomouc in 2009 and 2010. MATERIAL AND METHODS: Absolute gene expression of carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR1), lung-specific X protein (LUNX) and hepatocyte growth factor receptor (c-met) was determined in peripheral blood, bone marrow and pulmonary blood of 50 lung cancer patients using real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR). RESULTS: (1) The LUNX marker is specific and sensitive for MSD detection in lung cancer patients. (2) The CEA positivity for MSD in the bone marrow correlated significantly with histopathological grading (GI-GIII). (3) Higher expression of CEA and c-met was found in pulmonary blood of patients with hilar or mediastinal lymphadenopathy. (4) Higher expression of MSD markers (CEA in bone marrow, c-met in peripheral blood and LUNX in pulmonary blood) correlated with higher pTNM classification. CONCLUSION: Minimal systemic disease detection in lung cancer patients is technically feasible using sufficiently sensitive and specific markers for RT-PCR. Minimal systemic disease detection can be used to guide further systemic treatment. This theory must be validated in a larger group of patients and correlated with clinical data, especially with survival data.

COVID-19 associated coagulopathy: Mechanisms and host-directed treatment.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is associated with specific coagulopathy that frequently occurs during the different phases of coronavirus disease 2019 (COVID-19) and can result in thrombotic complications and/or death. This COVID-19-associated coagulopathy (CAC) exhibits some of the features associated with thrombotic microangiopathy, particularly complement-mediated hemolytic-uremic syndrome. In some cases, due to the anti-phospholipid antibodies, CAC resembles catastrophic anti-phospholipid syndrome. In other patients, it exhibits features of hemophagocytic syndrome. CAC is mainly identified by: increases in fibrinogen, D-dimers, and von Willebrand factor (released from activated endothelial cells), consumption of a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13 (ADAMTS13), over activated and dysregulated complement, and elevated plasma cytokine levels. CAC manifests as both major cardiovascular and/or cerebrovascular events and dysfunctional microcirculation, which leads to multiple organ damage. It is not clear whether the mainstay of COVID-19 is complement overactivation, cytokine/chemokine activation, or a combination of these activities. Available data have suggested that non-critically ill hospitalized patients should be administered full-dose heparin. In critically ill, full dose heparin treatment is discouraged due to higher mortality rate. In addition to anti-coagulation, four different host-directed therapeutic pathways have recently emerged that influence CAC: (1) Anti-von Willebrand factor monoclonal antibodies; (2) activated complement C5a inhibitors; (3) recombinant ADAMTS13; and (4) Interleukin (IL)-1 and IL-6 antibodies. Moreover, neutralizing monoclonal antibodies against the virus surface protein have been tested. However, the role of antiplatelet treatment remains unclear for patients with COVID-19.

[News in the classification of pulmonary adenocarcinomas and potential prognostic and predictive factors in non-small lung cancer]. / Novinky v klasifikaci adenokarcinomu plic a potenciální prognostické a prediktivní faktory u nemalobunecných plicních karcinomu.

Lung cancers are still divided into two major subgroups: small-cell and non-small cell lung cancer (NSCLC) irrespective of biological heterogeneity of NSCLC. It is a key task of the pathologist to provide an accurate classification of tumorous lesions to avoid the term NSCLC and to use it only in the vast minority of cases. Moreover, the most recent reclassification of pulmonary adenocarcinomas should be reflected in the standard biopsy protocol reporting. There is also an increasingly urgent need to provide high quality material for testing of the genetic characteristics of NSCLC, especially the presence and functional status of the EGFR receptor (epidermal growth factor receptor), as well as other potential prognostic markers. The requirement for the quality and swiftness of diagnosis puts major emphasis on the close multidisciplinary collaboration with the central role of a specialized pathologist, who coordinates the differential-diagnostic procedure. This in turn implies the necessity of accounting for the increasing financial burden of diagnostic departments.

LC3A positive "stone like structures" are differentially associated with survival outcomes and CD68 macrophage infiltration in patients with lung adenocarcinoma and squamous cell carcinoma.

AIMS: The aim of the study was to analyse the prognostic and predictive value of LC3A positive' 'Stone Like Structures'' (SLSs) in a large cohort of patients with non-small cell lung carcinoma (NSCLC) and to check its relationship with tumor infiltrating lymphocytes (TILs) and PD-L1 expression. METHODS: Tissue microarrays from 1015 patients diagnosed at the Institute of Pathology and Molecular Pathology, University Hospital Zurich, Switzerland, were stained for LC3A, PD-L1, CD3 and CD68 using automated tissue stainer Ventana Benchmark Ultra (Roche). TILs were assessed in matched haematoxylin and eosin stained slides. RESULTS: LC3A positive SLSs, were significantly associated with worse overall (OS) and disease-free survival (DFS) outcomes in patients with lung adenocarcinoma (LADC) (HR = 2.4, 95 %CI(.994-1.008, p = 0.029) and HR = 3.9, 95 %CI (1.002-1.014), p = 0.002 respectively), whilst it was associated with better OS and DFS in patients with lung squamous cell carcinoma (LUSC), with marginal significance (HR = .99, 95 %CI(.975-1.011),p = 0.042 and HR = .99, 95 %CI (.975-1.008), p = 0.026). Multivariate analysis showed that LC3A SLSs are independent poor prognostic factor only in patients with LADC. In addition, LC3A SLSs, were negatively associated with CD68 count in LADC, whilst there was a positive correlation in LSCC. CONCLUSIONS: LC3A SLSs are differentially associated with the survival outcomes and CD68 count in LADC and LSCC. Further studies are justified for the understanding the underlying biological mechanisms of this phenomenon.

[Prognostic impact of matrix metalloproteinases 2,9, and 11 in stromal cells stage I non-small cell lung cancer]. / Prognostický význam matrix metaloproteáz 2,9 a 11 ve stromáilních bunkách I. stadia nemalobunecného karcinomu plic.

BACKGROUND: Matrix metalloproteinases (MMPs) belong to proteolytic enzymes. Degradation of the cell basement membrane and the extracellular matrix is one of their functions. In malignant tumors they can hypothetically contribute to the invasion and metastasis formation. They are mostly produced by stromal cells (fibroblasts and endothelial cells) as a response to the presence of tumor cells. MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-11 (stromelysin 3) are often mentioned in regard to Non-small Cell Lung Cancer (NSCLC). METHODS AND RESULTS: The relation between the expression of the above-mentioned matrix metal-loproteinases in stromal cells and the cancer-related survival in 80 patients after curative resection of NSCLC in stage I according to TNM was studied. The expression of MMP-2 was associated with cancer-related survival but without significant correlation. No correlation was found in MMP-9. There was a statistically near-significant relation between the expression of MMP-11 and cancer-related survival. CONCLUSIONS: The expression of MMP-11 in stromal cells in surgically treated NSCLC patients in stage I appears useful for evaluation of their prognosis.

MMP-19 deficiency causes aggravation of colitis due to defects in innate immune cell function.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.

Histomorphometric diagnostics of renal osteopathy in chronic dialysis patients at high risk of cardiovascular disease.

UNLABELLED: Chronic kidney disease-mineral and bone disorder (CKD-MBD) ranks among clinically and pathogenetically significant complications in patients with CKD. Numerous factors are involved in its development, and histomorphometric analysis of the bone tissue is still necessary for accurate diagnosis. METHODS: The open, pilot, prospective study aimed at performing a comprehensive histomorphometric bone analysis in 26 dialysis patients and assessing the relationships of different types of CKD-MBD to selected parameters of calcium and phosphate metabolism, densitometry, activity of parathyroid glands, presence of diabetes mellitus, and duration of dialysis treatment. RESULTS: Comparison of the histomorphometric characteristics demonstrated statistically significant correlations between the volume of bone trabeculae and s-procollagen 1 (.754) as well as s-calcitonin (.856). Similarly, there was a positive correlation between the size of tetracycline lines and volume of bone trabeculae (.705) and a strong negative correlation with the thickness of trabeculae (-.442). When assessing the serum levels of s-osteoprotegerin and serum RANKL, there was a correlation with osteoid thickness and bone trabeculae thickness. In case of s-osteoprotegerin, a statistical power was demonstrated in relation to osteoid thickness (.880); in case of s-RANKL, a statistical power was demonstrated in relation to the thickness of trabeculae (.830). When assessing the influence of dialysis duration, relationships to the volume of trabecular bone (.665) and volume of bone trabeculae (.949) were demonstrated. Finally, a relationship between s-1,25-hydroxyvitamin D and s-osteoprotegerin was observed (.739); also the relationships demonstrated were significantly lower volume of bone trabeculae in men (p = 0.067) and lower values of s-osteocalcin and s-procollagen 1 in diabetic patients (p = 0.014). CONCLUSION: The results provide new noninvasive possibilities of CKD-MBD detection that are based on selected serum parameters of bone metabolism. Presented are possibilities of noninvasive assessment of different types of CKD-MBD using serum osteomarkers in relation to comprehensive CKD-MBD histomorphometry.

Growth hormone treatment increases oestrogen receptor concentration in the guinea-pig uterus.

Growth hormone does not act as a body growth-promoting hormone during the postnatal period in the guinea-pig. To determine whether it affects uterine and mammary growth, guinea-pigs of 8-9 weeks of age were treated with either recombinant bovine GH (bGH; 0.5 mg per animal) or vehicle for 10 days. Uterine and mammary weights were not changed by treating the animals with bGH. The amount of available cytosolic oestradiol receptor per unit of uterine weight or DNA content, or in whole uteri was increased in bGH-treated animals (7.3- to 14.0-fold) when compared with controls. The nuclear uterine oestradiol receptor concentration was 3.3- to 6.7-fold higher than in controls. The dissociation constant values did not differ between control and bGH-treated animals, suggesting that uterine oestradiol receptors are regulated by GH through changes in the number of binding sites rather than alteration of their binding affinity. Mammary growth and oestradiol receptor levels were unaffected by bGH treatment. The results of this investigation demonstrate that injected bGH selectively affects the oestradiol receptor level in guinea-pig uterine tissue.

Indications and results of liver resection and hepatic chemoembolization for metastatic gastrointestinal neuroendocrine tumors.

BACKGROUND: We reviewed 36 patients with liver metastases from islet cell tumors of the pancreas (n = 18) and carcinoid tumors (n = 18) who were treated with surgical resection (n = 16) or hepatic chemoembolization (n = 20). METHODS: All resections were complete and included 4 lobectomies, 6 segmental resections, and 6 wedge resections. There were no operative deaths. RESULTS: Median survival has not yet been reached, and the actuarial 5-year survival rate is 70%. Prognostic variables associated with improved disease-free survival included prior resection of the primary tumor and 4 or fewer metastases resected (P <.05). With an average of 3 chemoembolization procedures per patient, 17 of 20 patients (90%) demonstrated either a significant radiographic response (n = 5), stabilization of tumor mass (n = 2), or improvement of clinical symptoms (n = 10). Factors related to a sustained response (more then 1 year) included surgical resection of the primary tumor, 4 or more chemoembolization procedures, and liver metastases of 5 cm or smaller. Median survival after treatment was 32 months (range, 7-63 months), and the actuarial 5-year survival rate was 40%. CONCLUSIONS: Surgical resection of metastatic neuroendocrine tumors provides the best chance for extended survival. Chemoembolization effectively improves clinical symptoms and, in selected patients, may provide sustained tumor control.

Hormonal control of net glucose-stimulated lipogenesis during transition from brown to white adipose tissue in the goat.

Perinatal (1-2 days of age) and one-month-old (24-32 days of age) male goats were used to investigate the effect of age and long-term culture (24 h) of perirenal and omental adipose explants in the presence of insulin, cortisol and bovine somatotropin (alone or in different combinations) on net glucose-stimulated lipogenesis (NGSL, i.e. the rate of lipogenesis in the presence of glucose minus the rate of lipogenesis in the absence of glucose) in the absence and in the presence of catecholamines in acute incubations (2 h). Mean values of NGSL in both freshly prepared and cultured explants were consistently lower in perinatal than in one-month-old goats. Cortisol alone decreased and combinations of insulin plus cortisol increased NGSL in perirenal explants of one-month-old animals. When perirenal explants from these one-month-old goats were cultured in the presence of insulin plus cortisol plus bovine somatotropin, the rates of lipogenesis were lower than those in cultures with insulin plus cortisol. No such effects of these hormones were noted in omental explants of both perinatal and one-month-old animals. In freshly prepared perirenal and omental explants, the rates of NGSL were inhibited by isoprenaline in tissues of both groups of animals and by noradrenaline in omental tissues of animals of the older group only. The mean values of NGSL in cultured explants of perinatal animals were not affected by noradrenaline. Isoprenaline inhibited NGSL in omental but not in perirenal tissue. In older animals the rates of NGSL were decreased by both noradrenaline and isoprenaline in perirenal and omental adipose tissues. Isoprenaline was more effective than noradrenaline in perirenal adipose tissue.

Chronic effects of somatotropin treatment in vivo and in vitro on lipogenic activity of goat adipose tissue in a glucose-free buffer during acute incubation.

Young castrated male goats (n = 8) were used to investigate the effect of long-term treatment with recombinant methionyl bovine somatotropin in a sustained release vehicle (bST; 100 mg at seven-day intervals in a 147-day experiment) and chronic culture (24 h) of omental adipose tissue in the presence of various hormones on lipogenic responses to catecholamines during acute incubation (2 h) in a sodium acetate supplemented glucose-free buffer. The rate of fatty acid synthesis in freshly-prepared adipose explants was low and did not differ from those cultured in the absence of hormones for 24 h. Hormonal combination of insulin (17 nmol.l(-1)) plus cortisol (138 nmol.l(-1)) or insulin plus recombinant enterokinase linker bST (4.5 nmol.l(-1) increased lipogenesis (P<0.05). Further addition of bST or cortisol decreased lipogenesis significantly (P<0.05) in the controls but not significantly in bST-treated animals. Cultured explants from either control or bST-treated animals showed significant inhibition of lipogenesis by both norepinephrine (10 micromol.l(-1)) and isoprenaline (10 micromol.l(-1)). BST treatment in vivo did not increase the responsiveness of cultured explants to norepinephrine in vitro, however, the responsiveness to isoprenaline(inhibition of lipogenesis) was greater in bST-treated animals than in the controls.

Effect of somatotropin on adipose tissue net glucose-stimulated lipogenesis in young goats.

Net glucose-stimulated lipogenesis (NGSL: the rate of lipogenesis in the presence of glucose minus the rate of lipogenesis in the absence of glucose) in omental adipose tissue explants from young castrated male goats was evaluated in control animals (n = 3; placebo-treated) and in animals treated with the sustained release of recombinant bovine somatotropin (n = 4; bST; 100 mg at 7-day intervals in a 147 days lasting experiment). The rate of fatty acid synthesis was determined in acute incubations in both freshly prepared and chronically cultured explants. Adipose explants remained metabolically active and retained their ability to respond to hormones when maintained in a tissue culture medium. NGSL in explants cultured for 24 h in the presence of insulin alone or bST alone, was non-significantly increased (more in the controls) and decreased (more in bST-treated animals), respectively. However, cortisol alone decreased (P<0.05) NGSL in explants from both control and bST-treated animals. In tissues from bST-treated animals, cortisol acted synergistically with insulin to produce a higher rate of NGSL than that observed in cultures with insulin alone. bST inhibited insulin plus cortisol-stimulated lipogenesis significantly (P<0.05) in explants from bST-treated animals but non-significantly in control animals. The rates of NGSL were decreased (P<0.05) by catecholamines in explants from both control and bST-treated animals. Norepinephrine (NE) and isoprenaline (ISO) were equally effective in the controls, whereas isoprenaline was more effective than norepinephrine in bST-treated animals.

Detection of estrogenicity by bioassay on the mouse mammary gland in vivo.

The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure or radioimmunological assay and thus requires that the potency of these compounds is defined by bioassay. The mammary duct growth response in intact prepubertal and adult gonadectomized female and male mice of the C3H/Di strain was used to assess the estrogenicity of synthetic compounds or their derivatives. The vehicle for tested compounds should be free of estrogenic and other hormonal effects. Olive oil or sunflower oil exerted estrogenic activities and were thus unsuitable as vehicles for the tested compounds. The absence of estrogenic activity, high solubility of different steroid hormones, and the low incidence of the inflammatory reactions at the injection site were achieved by using a vehicle containing benzyl alcohol, benzyl benzoate, butylhydroxyanisole, butylhydroxytoluene, ethyl oleate and ethanol. The bioassay was primarily designed to examine the effect of tested compounds on mammogenesis. The duration of hormone treatment was chosen to be long enough for induction of duct growth but too short to induce lobuloalveolar differentiation. Females were treated for 10 days, males for 15 days. The proportional volume occupied by mammary epithelial structures was estimated by the modified Chalkley's technique. The mean coefficient of variation of quantitative evaluation of 10 different mammary glands obtained by two operators varied between 3.2 and 17.4%. The mean coefficient of variation of quintuplicate determinations of each mammary gland by one operator was 10.1%, and 11.1% by the other. The correlation coefficient between results of two operators was 0.994. Estrogens are primarily defined by their ability to increase the mitotic activity of female secondary sex organs. However, our results have shown that progesterone alone, if administered in a high dose, stimulates mammary growth in both intact prepubertal and OV-X female mice similarly as the synthetic progestatial steroid norethindrone with inherent estrogenic properties. In contrast, progesterone alone had no effect, in young intact or adult castrated males, but norethindrone did stimulate mammary growth. These results demonstrated that the mammary gland of males is a suitable model for estrogen screening.

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice.

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice.

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.