Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Dose-dependent effects of 17-ß-estradiol on pituitary thyrotropin content and secretion in vitro

We studied the basal and thyrotropin-releasing hormone (TRH) (50 nM) induced thyrotropin (TSH) release in isolated hemipituitaries of ovariectomized rats treated with near-physiological or high doses of 17-ß-estradiol benzoate (EB; sc, daily for 10 days) or with vehicle (untreated control rats, OVX). One group was sham-operated (normal control). The anterior pituitary glands were incubated in Krebs-Ringer bicarbonate medium, pH 7.4, at 37oC in an atmosphere of 95% O2/5% CO2. Medium and pituitary TSH was measured by specific RIA (NIDDK-RP-3). Ovariectomy induced a decrease (P<0.05) in basal TSH release (normal control = 44.1 ± 7.2; OVX = 14.7 ± 3.0 ng/ml) and tended to reduce TRH-stimulated TSH release (normal control = 33.0 ± 8.1; OVX = 16.6 ± 2.4 ng/ml). The lowest dose of EB (0.7 µg/100 g body weight) did not reverse this alteration, but markedly increased the pituitary TSH content (0.6 ± 0.06 µg/hemipituitary; P<0.05) above that of OVX (0.4 ± 0.03 µg/hemipituitary) and normal rats (0.46 ± 0.03 µg/hemipituitary). The intermediate EB dose (1.4 µg/100 g body weight) induced a nonsignificant tendency to a higher TSH response to TRH compared to OVX and a lower response compared to normal rats. Conversely, in the rats treated with the highest dose (14 µg/100 g body weight), serum 17-ß-estradiol was 17 times higher than normal, and the basal and TRH-stimulated TSH release, as well as the pituitary TSH content, was significantly (P<0.05) reduced compared to normal rats and tended to be even lower than the values observed for the vehicle-treated OVX group, suggesting an inhibitory effect of hyperestrogenism. In conclusion, while reinforcing the concept of a positive physiological regulatory role of estradiol on the TSH response to TRH and on the pituitary stores of the hormone, the present results suggest an inhibitory effect of high levels of estrogen on these responses

Paraquat poisoning: an experimental model of dose-dependent acute lung injury due to surfactant dysfunction

Since the most characteristic feature of paraquat poisoning is lung damage, a prospective controlled study was performed on excised rat lungs in order to estimate the intensity of lesion after different doses. Twenty-five male, 2-3-month-old non-SPF Wistar rats, divided into 5 groups, received paraquat dichloride in a single intraperitoneal injection (0, 1, 5, 25, or 50 mg/kg body weight) 24 h before the experiment. Static pressure-volume (PV) curves were performed in air- and saline-filled lungs; an estimator of surface tension and tissue works was computed by integrating the area of both curves and reported as work/ml of volume displacement. Paraquat induced a dose-dependent increase of inspiratory surface tension work that reached a significant two-fold order of magnitude for 25 and 50 mg/kg body weight (P<0.05, ANOVA), sparing lung tissue. This kind of lesion was probably due to functional abnormalities of the surfactant system, as was shown by the increase in the hysteresis of the paraquat groups at the highest doses. Hence, paraquat poisoning provides a suitable model of acute lung injury with alveolar instability that can be easily used in experimental protocols of mechanical ventilation

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of ~65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178 ® stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, I detected ~110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing - structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.

Influence of dexamethasone and weight loss on the regulation of serum leptin levels in obese individuals

The adipocyte hormone leptin is thought to serve as a signal to the central nervous system reflecting the status of fat stores. Serum leptin levels and adipocyte leptin messenger RNA levels are clearly increased in obesity. Nevertheless, the factors regulating leptin production are not fully understood. The aim of this study was to determine the effects of in vivo administration of the synthetic glucocorticoid dexamethasone and weight loss on serum leptin levels in two independent protocols. Twenty-five obese subjects were studied (18 women and 7 men, mean age 26.6 ± 6 years, BMI 31.1 ± 2.5 kg/m², %fat 40.3 ± 8.3) and compared at baseline to 22 healthy individuals. Serum levels of leptin, insulin, proinsulin and glucose were assessed at baseline and after ingestion of dexamethasone, 4 mg per day (2 mg, twice daily) for two consecutive days. To study the effects of weight loss on serum leptin, 17 of the obese subjects were submitted to a low-calorie dietary intervention trial for 8 weeks and again blood samples were collected. Serum leptin levels were significantly higher in the obese group compared to the control group and a high positive correlation between leptinemia and the magnitude of fat mass was found (r = 0.88, P<0.0001). After dexamethasone, there was a significant increase in serum leptin levels (22.9 ± 12.3 vs 51.4 ± 23.3 ng/ml, P<0.05). Weight loss (86.1 ± 15.1 vs 80.6 ± 14.2 kg, P<0.05) led to a reduction in leptin levels (25.13 ± 12.8 vs 15.9 ± 9.1 ng/ml, P<0.05). We conclude that serum leptin levels are primordially dependent on fat mass magnitude. Glucocorticoids at supraphysiologic levels are potent secretagogues of leptin in obese subjects and a mild fat mass reduction leads to a disproportionate decrease in serum leptin levels. This suggests that, in addition to the changes in fat mass, complex nutritional and hormonal interactions may also play an important role in the regulation of leptin levels.

Combined effect of short stature and socioeconomic status on body mass index and weight gain during reproductive age in Brazilian women

Short stature, a marker for undernutrition early in life, has been associated with obesity in Brazilian women, but not in men. We tested the hypothesis that weight gain during the reproductive years could explain this gender difference. A national two-stage household survey of mothers with one or more children under five years of age was conducted in Brazil in 1996. The subjects were women aged 20 to 45 years (N = 2297), with last delivery seven months or more prior to the interview. The regions of the country were divided into rural, North/Northeast (urban underdeveloped) and South/Southeast/Midwest (urban developed). The dependent variables were current body mass index (BMI) measured, BMI prior to childbearing (reported), and BMI change. Socioeconomic variables included mother's years of education and family purchasing power score. A secondary analysis was restricted to primiparous women. The prevalence of current overweight and overweight prior to childbearing (BMI > or = 25 kg/m²) was higher among shorter women (<1.50 m) compared to normal stature women only in the urban developed region (P < 0.05). After adjustment for socioeconomic variables, age, parity, BMI prior to childbearing, and age at first birth, current BMI was 2.39 units higher (P = 0.008) for short stature women living in the urban developed area compared with short stature women living in the urban underdeveloped area. For both multiparous and primiparous women, BMI gain compared to the value prior to childbearing was significantly higher among short stature women living in the urban developed region (P <= 0.04). These results provide clear evidence that short stature was associated with a higher BMI and with an increased risk of weight gain/retention with pregnancy in the developed areas of Brazil, but not in the underdeveloped ones.

Chronic experimental myocardial infarction produces antinatriuresis by a renal nerve-dependent mechanism

The present study focused on the role of sympathetic renal nerve activity, in mediating congestive heart failure-induced sodium retention following experimental chronic myocardial infarction. Groups of male Wistar rats (240-260 g) were studied: sham-operated coronary ligation (CON3W, N = 11), coronary ligation and sham-operated renal denervation (INF3W, N = 19), 3 weeks of coronary ligation and sympathetic renal nerve denervation (INF3WDX, N = 6), sham-operated coronary ligation (N = 7), and 16 weeks of coronary ligation (INF16W, N = 7). An acute experimental protocol was used in which the volume overload (VO; 5% of body weight) was applied for 30 min after the equilibration period of continuous iv infusion of saline. Compared to control levels, VO produced an increase (P < 0.01, ANOVA) in urine flow rate (UFR; 570%) and urinary sodium excretion (USE; 1117%) in CON3W. VO induced a smaller increase (P < 0.01) in USE (684%) in INF3W. A similar response was also observed in INF16W. In INF3WDX, VO produced an immediate and large increase (P < 0.01) in UFR (547%) and USE (1211%). Similarly, in INF3W VO increased (P < 0.01) UFR (394%) and USE (894%). Compared with INF3W, VO induced a higher (P < 0.01) USE in INF3WDX, whose values were similar to those for CON3W. These results suggest that renal sympathetic activity may be involved in sodium retention induced by congestive heart failure. This premise is supported by the observation that in bilaterally renal denervated INF3WDX rats myocardial infarction was unable to reduce volume expansion-induced natriuresis. However, the mechanism involved in urinary volume regulation seems to be insensitive to the factors that alter natriuresis.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Computerized invasive measurement of time-dependent intraocular pressure

Several methods have been described to measure intraocular pressure (IOP) in clinical and research situations. However, the measurement of time varying IOP with high accuracy, mainly in situations that alter corneal properties, has not been reported until now. The present report describes a computerized system capable of recording the transitory variability of IOP, which is sufficiently sensitive to reliably measure ocular pulse peak-to-peak values. We also describe its characteristics and discuss its applicability to research and clinical studies. The device consists of a pressure transducer, a signal conditioning unit and an analog-to-digital converter coupled to a video acquisition board. A modified Cairns trabeculectomy was performed in 9 Oryctolagus cuniculus rabbits to obtain changes in IOP decay parameters and to evaluate the utility and sensitivity of the recording system. The device was effective for the study of kinetic parameters of IOP, such as decay pattern and ocular pulse waves due to cardiac and respiratory cycle rhythm. In addition, there was a significant increase of IOP versus time curve derivative when pre- and post-trabeculectomy recordings were compared. The present procedure excludes corneal thickness and error related to individual operator ability. Clinical complications due to saline infusion and pressure overload were not observed during biomicroscopic evaluation. Among the disadvantages of the procedure are the requirement of anesthesia and the use in acute recordings rather than chronic protocols. Finally, the method described may provide a reliable alternative for the study of ocular pressure dynamic alterations in man and may facilitate the investigation of the pathogenesis of glaucoma.

Streptozotocin-induced mechanical hypernociception is not dependent on hyperglycemia

Since streptozotocin (STZ)-induced diabetes is a widely used model of painful diabetic neuropathy, the aim of the present study was to design a rational protocol to investigate whether the development of mechanical hypernociception induced by STZ depends exclusively on hyperglycemia. Male Wistar rats (180-200 g; N = 6-7 per group) received a single intravenous injection of STZ at three different doses (10, 20, or 40 mg/kg). Only the higher dose (40 mg/kg) induced a significant increase in blood glucose levels, glucose tolerance and deficiency in weight gain. However, all STZ-treated rats (hyperglycemic or not) developed persistent (for at least 20 days) and indistinguishable bilateral mechanical hypernociception that was not prevented by daily insulin treatment (2 IU twice a day, sc). Systemic morphine (2 mg/kg) but not local (intraplantar) morphine treatment (8 µg/paw) significantly inhibited the mechanical hypernociception induced by STZ (10 or 40 mg/kg). In addition, intraplantar injection of STZ at doses that did not cause hyperglycemia (30, 100 or 300 µg/paw) induced ipsilateral mechanical hypernociception for at least 8 h that was inhibited by local and systemic morphine treatment (8 µg/paw or 2 mg/kg, respectively), but not by dexamethasone (1 mg/kg, sc). The results of this study demonstrate that systemic administration of STZ induces mechanical hypernociception that does not depend on hyperglycemia and intraplantar STZ induces mechanical sensitization of primary sensory neurons responsive to local morphine treatment.

L-histidine provokes a state-dependent memory retrieval deficit in mice re-exposed to the elevated plus-maze

The effects of L-histidine (LH) on anxiety and memory retrieval were investigated in adult male Swiss Albino mice (weight 30-35 g) using the elevated plus-maze. The test was performed on two consecutive days: trial 1 (T1) and trial 2 (T2). In T1, mice received an intraperitoneal injection of saline (SAL) or LH before the test and were then injected again and retested 24 h later. LH had no effect on anxiety at the dose of 200 mg/kg since there was no difference between the SAL-SAL and LH-LH groups at T1 regarding open-arm entries (OAE) and open-arm time (OAT) (mean ± SEM; OAE: 4.0 ± 0.71, 4.80 ± 1.05; OAT: 40.55 ± 9.90, 51.55 ± 12.10, respectively; P > 0.05, Kruskal-Wallis test), or at the dose of 500 mg/kg (OAE: 5.27 ± 0.73, 4.87 ± 0.66; OAT: 63.93 ± 11.72, 63.58 ± 10.22; P > 0.05, Fisher LSD test). At T2, LH-LH animals did not reduce open-arm activity (OAE and OAT) at the dose of 200 mg/kg (T1: 4.87 ± 0.66, T2: 5.47 ± 1.05; T1: 63.58 ± 10.22; T2: 49.01 ± 8.43 for OAE and OAT, respectively; P > 0.05, Wilcoxon test) or at the dose of 500 mg/kg (T1: 4.80 ± 1.60, T2: 4.70 ± 1.04; T1: 51.55 ± 12.10, T2: 43.88 ± 10.64 for OAE and OAT, respectively; P > 0.05, Fisher LSD test), showing an inability to evoke memory 24 h later. These data suggest that LH does not act on anxiety but does induce a state-dependent memory retrieval deficit in mice.

Physical training prevents body weight gain but does not modify adipose tissue gene expression

The relationship of body weight (BW) with white adipose tissue (WAT) mass and WAT gene expression pattern was investigated in mice submitted to physical training (PT). Adult male C57BL/6 mice were submitted to two 1.5-h daily swimming sessions (T, N = 18), 5 days/week for 4 weeks or maintained sedentary (S, N = 15). Citrate synthase activity increased significantly in the T group (P < 0.05). S mice had a substantial weight gain compared to T mice (4.06 ± 0.43 vs 0.38 ± 0.28 g, P < 0.01). WAT mass, adipocyte size, and the weights of gastrocnemius and soleus muscles, lung, kidney, and adrenal gland were not different. Liver and heart were larger and the spleen was smaller in T compared to S mice (P < 0.05). Food intake was higher in T than S mice (4.7 ± 0.2 vs 4.0 ± 0.3 g/animal, P < 0.05) but oxygen consumption at rest did not differ between groups. T animals showed higher serum leptin concentration compared to S animals (6.37 ± 0.5 vs 3.11 ± 0.12 ng/mL). WAT gene expression pattern obtained by transcription factor adipocyte determination and differentiation-dependent factor 1, fatty acid synthase, malic enzyme, hormone-sensitive lipase, adipocyte lipid binding protein, leptin, and adiponectin did not differ significantly between groups. Collectively, our results showed that PT prevents BW gain and maintains WAT mass due to an increase in food intake and unchanged resting metabolic rate. These responses are closely related to unchanged WAT gene expression patterns.

Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway

This study investigated the in vitro and in vivo antiproliferative activity of esculetin against hepatocellular carcinoma, and clarified its potential molecular mechanisms. Cell viability was determined by the MTT (tetrazolium) colorimetric assay. In vivoantitumor activity of esculetin was evaluated in a hepatocellular carcinoma mouse model. Seventy-five C57BL/6J mice were implanted with Hepa1-6 cells and randomized into five groups (n=15 each) given daily intraperitoneal injections of vehicle (physiological saline), esculetin (200, 400, or 700 mg·kg-1·day-1), or 5-Fu (200 mg·kg-1·day-1) for 15 days. Esculetin significantly decreased tumor growth in mice bearing Hepa1-6 cells. Tumor weight was decreased by 20.33, 40.37, and 55.42% with increasing doses of esculetin. Esculetin significantly inhibited proliferation of HCC cells in a concentration- and time-dependent manner and with an IC50 value of 2.24 mM. It blocked the cell cycle at S phase and induced apoptosis in SMMC-7721 cells with significant elevation of caspase-3 and caspase-9 activity, but did not affect caspase-8 activity. Moreover, esculetin treatment resulted in the collapse of mitochondrial membrane potential in vitro and in vivo accompanied by increased Bax expression and decreased Bcl-2 expression at both transcriptional and translational levels. Thus, esculetin exerted in vitro and in vivo antiproliferative activity in hepatocellular carcinoma, and its mechanisms involved initiation of a mitochondrial-mediated, caspase-dependent apoptosis pathway.

Secretory immunity in overweight and obese versus normal-weight early-and late-pubertal females

Obesity is a condition associated with a wide variety of health problems including hypertension, dyslipidemia, diabetes mellitus, certain forms of cancer, cardiovascular disease, and gallstones (157). TTiere is growing evidence that obesity may also be related to compromised immune function due to altered metabolic, psychological, and physical attributes (93). The aim of this study was to compare: a) immunity-related variables such as frequency of upper respiratory tract infections (URTI) and salivary secretory immunoglobulin A (sIgA) levels between overweight/obese (OB) and normal weight (NW) early-pubertal and late-pubertal girls, and b) stress-related variables such as Cortisol, melatonin, the melatonin/cortisol ratio, testosterone and the testosterone/cortisol ratio. Physical activity levels, stress indicators, and fatigue were used to explain potential differences in the dependent variables. It was hypothesized that the OB females would have lower melatonin (M) and higher Cortisol (C) and testosterone (T) levels compared with NW girls, regardless of maturity status. The altered levels of melatonin, Cortisol, and testosterone, would result in decreased M/C and T/C ratios, despite the increase in testosterone in OB females. It was hypothesized that this altered hormonal status results in a compromised immunity marked by higher frequency of upper respiratory tract infections (URTI) and decreased levels of secretory immunoglobulin A (sIgA). It was also hypothesized that OB girls would participate in less hours of physical activity than their NW counterparts and that this would relate to their stress and immunity levels. Forty (16 early- and 24 late-pubertal) overweight and obese females were compared to fifty-three (27 eariy- and 26 late-pubertal) age-matched normal-weight control subjects. Participants were categorized as early-pubertal (EP) or late-pubertal (LP) using Tanner self-staging of secondary sex characteristics. Subjects were classified into the two adiposity groups according to relative body fat (%BF), where normal weight (NW) subjects had a %BF less than 25%, and overweight and obese (OB) subjects had a %BF greater than 27.5%. Participants completed a number of questionnaires and information was collected on menstrual history, smoking history, alcohol and caffeine consumption, and medical history. Following the determination of maturity status, a complete anthropometric assessment was made including height, body mass, and body composition. All questionnaires and measurements were completed during a one-hour visit between 1 500 and 1900 hours Relative body fat was assessed using bioelectrical impedance analysis. Resting saliva samples were obtained and assayed (ELISA) for testosterone, Cortisol, melatonin and secretory immunoglobulin A. Physical activity was self-reported using the Godin- Shephard Leisure time questionnaire, and quantified using Actigraph GTIM accelerometers, which participants wore for seven consecutive days from the time they woke up in the morning, until the time they went to bed. Late-pubertal girls also completed questionnaires on their perceived stress and fatigue. Finally, all participants also filled out a one-month health log to record frequency of symptoms of upper respiratory tract infections (URTI). Significant age effects were found for testosterone, Cortisol, incidence of sickness, and sIgA when controlling for physical activity, however there were no significant effects of adiposity on any of the variables. There was a trend which neared-significance for an effect of adiposity on sIgA (p=0.01). There were no significant differences between the groups on the total selfreported leisure-time physical activity in METs per week, however EP girls recorded significantly greater levels of moderate, hard, and very hard physical activity from accelerometers. Results of the perceived stress and fatigue questionnaires in late-pubertal girls demonstrated that contrary to what was hypothesized, NW girls reported more stress and more fatigue than OB girls. Results of the present study suggest that excess adiposity in early- and latepubescent girls may not have a negative impact on immunity as hypothesized.