Cross-border reproductive care use by women with infertility in Hong Kong: cross-sectional survey.

OBJECTIVES: Cross-border reproductive care (CBRC) is an increasingly common global phenomenon, but there is a lack of information regarding its frequency among residents of Hong Kong. This study aimed to evaluate the use of CBRC and the factors affecting its use among residents of Hong Kong. METHODS: This cross-sectional questionnaire study collected data from 1204 women with infertility who attended Hong Kong Hospital Authority and Family Planning Association infertility clinics. RESULTS: In total, 178 women (14.8% of all respondents) had used CBRC. Among respondents who had not used CBRC, 36.3% planned to use or would consider it. The main factors influencing the likelihood of using CBRC among women with infertility in Hong Kong use were long waiting times in the public sector and high cost in the private sector. Taiwan was the most preferred destination for CBRC (69.6% of respondents). Most information concerning CBRC was accessed via the internet. More than two thirds of respondents believed that the government in Hong Kong should formulate some regulations or guidance regarding CBRC. CONCLUSION: Nearly one in six women with infertility in Hong Kong had used CBRC. Among women who had not used CBRC, more than one third planned to use or would consider it. The main factors influencing the likelihood of CBRC use were long waiting times in the public sector and high cost in the private sector. These results will help clinicians to more effectively counsel patients considering CBRC and facilitate infertility services planning by authorities in Hong Kong.

Unraveling the adipocyte inflammomodulatory pathways activated by North American ginseng.

BACKGROUND: North American (NA) ginseng (Panax quinquefolius) is a popular natural health product (NHP) that has been demonstrated to regulate immune function, inflammatory processes and response to stress and fatigue. Recent evidence suggests that various extracts of NA ginseng may have different bioactivities because of distinct profiles of ginsenosides and polysaccharides. To date, the bioactive role of ginseng on adipocytes remains relatively unexplored. OBJECTIVE: The goal of this work was to study the extract-specific bioactivity of NA ginseng on differentiated preadipocyte gene expression and adipocytokine secretion. METHODS: In vitro differentiated 3T3-L1 preadipocytes were treated with 25 and 50 µg ml of either crude ethanol (EtOH) or aqueous (AQ) NA ginseng extracts, or polysaccharide and ginsenoside extracts isolated from the AQ extract. Global gene expression was studied with microarrays and the resulting data were analyzed with functional pathway analysis. Adipocytokine secretion was also measured in media. RESULTS: Pathway analysis indicated that the AQ extract, and in particular the polysaccharide extract, triggered a global inflammomodulatory response in differentiated preadipocytes. Specifically, the expression of Il-6 (interleukin 6), Ccl5 (chemokine (C-C motif) ligand 5), Nfκb (nuclear factor-kappaB) and Tnfα (tumor necrosis factor alpha) was increased. These effects were also reflected at the protein level through the increased secretion of IL-6 and CCL5. No effect was seen with the EtOH extract or ginsenoside extract. Using a specific toll-like receptor 4 (TLR4) inhibitor reduced the upregulation of inflammatory gene expression, indicating the relevance of this pathway for the signaling capacity of NA ginseng polysaccharides. CONCLUSION: This work emphasizes the distinct bioactivities of different ginseng extracts on differentiated preadipocyte signaling pathways, and highlights the importance of TLR4 for mediating the inflammomodulatory role of ginseng polysaccharides.

Effects of long-chain fatty amines on the growth of ras-transformed NIH 3T3 cells.

A number of aliphatic primary amines were tested for their effects on the growth of ras-transformed NIH 3T3 cells (PAP2 cells), as measured by incorporation of tritiated thymidine into DNA. Long-chain, saturated amines (C12 to C18) were growth inhibitory, whereas short-chain amines (C6, C8) were not. Farnesylamine, a branched-chain, unsaturated amine (C15), had an IC50 of 6.9 microM compared to IC50 values of 13.1 to 45.8 microM for straight-chain, saturated amines. Oleylamine, with an IC50 of 0.1 microM, was the most potent inhibitor. The long-chain amines, but not the short-chain amines, were also effective inhibitors of protein kinase C, assayed in vitro in a cell-free system. In addition, studies with indo-1-loaded PAP2 cells showed that long-chain amines induced a reversible rise in intracellular free Ca2+ concentration. Growth inhibition by the amines was positively correlated with this effect, suggesting that factors other than protein kinase C may be involved in the inhibition of growth of PAP2 cells by long-chain amines.

Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages.

A concentration-dependent elevation of intracellular calcium ([Ca2+]i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (< or = 100 microM in 1 ml with 1 x 10(6) cells). Oxidation of NAD(P)H preceded a rise in [Ca2+]i. The elevation of [Ca2+]i was reversible at < 50 microM tBOOH exposure and the return to the steady state [Ca2+]i correlated temporally with repletion of NAD(P)H. At > 50 microM tBOOH, the changes in NAD(P)H and [Ca2+]i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at < 50 microM tBOOH, oxidation of NADPH predominated, while at > 50 microM tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca2+]i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca2+]i at < 50 microM tBOOH; (ii) the sustained elevation of [Ca2+]i at > 50 microM tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca2+]i did not depend on influx of extracellular Ca2+. We speculate that at low tBOOH, Ca2+ was released from the NADPH/NADP(+)-sensitive mitochondrial Ca2+ pool while higher tBOOH caused additional Ca2+ release from GSH/GSSG-sensitive nonmitochondrial Ca2+ pools with sustained elevation of [Ca2+]i due to decreased mitochondrial Ca2+ reuptake.

Developmental profiles of antioxidant enzymes and trace metals in chick embryo.

It has been previously well documented that partial pressure of oxygen (PO2) and weight-specific rate of O2 consumption in chick embryo (Gallus gallus domesticus) transiently increase midway through the 21-day in ovo incubation period. The present study found that these oxidative changes were paralleled by the concentrations of glutathione (GSH) and Zn in liver and by the specific activity of superoxide dismutase (SOD) in brain. Levels of antioxidant enzymes and their trace metal cofactors were markedly higher in liver than in brain. Hepatic catalase activity changed in parallel with the concentration of its cofactor, Fe. However, the relative abundance of metal cofactors did not appear to be the determining influence on other antioxidant enzyme activities. Rates of extra-mitochondrial hydrogen peroxide release were also much greater in liver than in brain. Taken together, the results of this initial study of embryonic chick antioxidant systems suggest that certain antioxidants may be regulated by PO2 and rate of oxidative metabolism during fetal development.

Age-related differences in iron-nitrilotriacetate hepatotoxicity in the guinea pig: role of copper metallothionein.

This study was concerned with the role of Cu and Cu-MT (metallothionein) in oxidative stress. Because hepatic Cu and Cu-MT concentrations are known to be high in the 3-day-old guinea pigs but decline to low adult levels by 7 days of life, the hepatotoxicity of ferric nitrilotriacetate (FeNTA) in the developing guinea pig was used as the experimental model in the present study. Results of this study showed that the hepatotoxic response to FeNTA (3.5 mg Fe /kg i.p.) as measured by elevation in serum aspartate aminotransferase activity, increase in lipid peroxidation, decrease in reduced glutathione/oxidized glutathione ratio and histopathological changes was higher in 3-day-old than in 7-day-old and adult guinea pigs. Furthermore, pretreatment of 7-day-old guinea pigs with cupric sulfate (0.5 mg Cu++/kg i.p.) increased hepatic Cu and Cu-MT levels and enhanced susceptibility to FeNTA. FeNTA treatment resulted in the oxidation of MT thiolates and reduction in the metal binding capacity and Cu content of MT in the 3-day-old and Cu-pretreated 7-day-old animals, providing evidence for the interaction between Cu-MT and cellular oxidants. In vitro study with FeNTA and hepatic microsomes revealed no age-related differences in microsomal lipid peroxidation; however, this parameter was stimulated in the presence of control or heat-treated cytosols isolated from 3-day-old but not those of 7-day-old animals. These observations were consistent with the involvement of Cu-MT, a heat-stable metalloprotein, in the sensitization of hepatic tissues to oxidative injury in the 3-day-old animal. Moreover, in vitro study involving the use of D-penicillamine, a Cu chelating agent, showed that the sensitization effect of Cu-MT was mediated by Cu ions. The results of this study suggest that Cu-MT may have a prooxidative property and tissues with high Cu-MT levels may be particularly susceptible to oxidative stress.

Biochemical mechanism of metallothionein--carbon tetrachloride interaction in vitro.

To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCl4) toxicity, in vitro experiments were carried out to study the interaction of metallothionein and CCl4. Results from this study showed that incubation of Cd,Zn-MT with CCl4 in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CCl4-induced decrease in MT-thiol content was due to the scavenging of CCl4 metabolite(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCl4-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of 14CCl4 to MT was detected following incubation in the presence of a microsomal bioactivation system, it did not account for the CCl4-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CCl4 on MT thiol content. Measurement of the thiol content of CCl4-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CCl4 treatment could be regenerated. These data suggest that CCl4-linked oxidation of MT, rather than the covalent binding of 14CCl4 metabolite(s), may be responsible for the CCl4-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However, the precise role of the metal released during the oxidation of MT in CCl4 toxicity remains to be defined.

Reproductive tract defects induced in adult male rats by postnatal 1,2-dibromo-3-chloropropane exposure.

The reproductive tract of the male rat may be particularly susceptible to chemical injury during the early postnatal period since significant developmental changes occur in the tract at that time. The subcutaneous administration of relatively low doses of 1,2-dibromo-3-chloropropane (DBCP, 5-20 mg/kg) on alternate days, from 2 to 20 days of life, resulted in a marked dose-related reduction in the testis, epididymis, and seminal vesicle weights. Histological evaluation revealed degenerative cellular changes in the testes of the 5 mg/kg treated group and obliteration of the seminiferous tubules in the 10 mg/kg treated animals. Biochemical studies showed that the in vitro androgen production capacity per unit weight of testicular tissue was elevated, as a function of the DBCP dose, correlating with the apparent increases in the Leydig cell concentrations observed histologically in the treated animals. Due to the marked reduction in the testes weight of DBCP-treated animals, the in vitro testicular androgen production rate, when expressed on the basis of testes pair weight, was reduced; it was consistent with the observed DBCP-induced decrease in serum androgen levels. Early DBCP exposure also obliterated the androgen responsiveness of the seminal vesicle and epididymis in the adult rat, which may also contribute to the diminution in the weights of these androgen-dependent organs. The present study also indicated that immature rats were more susceptible than sexually matured male rats to DBCP toxicity. Moreover, the results of the critical period study indicated that the first 10 days of life are of particular importance in the reproductive tract toxicity of DBCP.

Metabolism of copper and zinc in the liver and bone of the perinatal guinea-pig.

Hepatic copper concentration in the guinea-pig increased markedly during the second-half of gestation, attaining a maximum shortly after birth; thereafter, concentration declined rapidly during the neonatal period. Changes in perinatal hepatic copper concentrations paralleled the binding of copper to a cytosolic metallothionein-like component, and the loss of hepatic copper in the neonates coincided with increases in serum copper concentrations. Zinc concentrations of the perinatal liver were low and showed no dramatic developmental changes. The humerus showed striking increases in zinc concentration with gestational age, attaining peak concentration before term and a marked depletion of tissue zinc during the neonatal period.

Effects of dexamethasone injection on body retention and hepatic distribution of zinc, cadmium and metallothionein in newborn rats.

The liver of 7-day-old rats contains high levels of metallothionein (MT) and zinc, which can be reduced markedly following treatment of neonates with dexamethasone (1 mg/kg, s.c.) twice daily on days 3, 4, 5 and 6 postpartum. Prior treatment with trace amounts of 65Zn and 109Cd did neither alter the basal MT and zinc concentrations nor their response to dexamethasone. The turnover of 65Zn or 109Cd in newborn rats was studied by measuring the whole body retention of the radioisotopes. Between days 3 and 7 postpartum, the normal turnover of 65Zn was much faster than that of 109Cd, and dexamethasone treatment resulted in further decrease of 65Zn retention without significantly altering that of 109Cd. The data indicate that dexamethasone enhanced the excretion of 65Zn without any effect on 109Cd excretion. The dexamethasone treatment resulted in marked reduction in both hepatic Zn and MT levels in newborn rats. Gel filtration on Sephadex G-75 columns of hepatic cytosols isolated from 7-day-old rats showed significant amounts of total Zn and 65Zn bound to MT fraction. Moreover, dexamethasone treatment markedly reduced their binding to the MT fraction suggesting that most of the Zn lost from the liver of dexamethasone-treated animals was from MT. About 95% of 109Cd in the liver cytosol was present in the MT fraction of 109Cd-injected rats and dexamethasone had little effect on the binding of 109Cd to MT fraction despite a marked reduction in the binding of total Zn. These data suggest that unlike in adult rats, injection of dexamethasone results in marked decrease in hepatic levels of Zn and MT in neonatal rats.

Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity.

To study the influence of hepatic metallothionein (MT) on the hepatotoxic response to carbon tetrachloride (CCl4), adult male rats were pretreated with a 10 mg X kg-1 dose of zinc (Zn) 24 h prior to CCl4 (i.p., l mL X kg-1) treatment. Zn pretreatment increased the hepatic MT concentrations markedly and reduced the magnitudes of the CCl4-induced reduction of cytochrome P450 concentration as well as elevation of serum alanine aminotransferase and aspartate aminotransferase activities when determined at 4 or 24 h following CCl4 treatment. Treatment of Zn-exposed animals with CCl4 also resulted in significant reduction of the concentrations of hepatic MT (as determined by the cadmium-saturation method) as well as cytosolic Zn. Sephadex G-75 chromatographic study of hepatic cytosols showed that MT-bound Zn was selectively depleted by CCl4 exposure. Moreover, it was demonstrated that CCl4, after metabolic activation, reduced the cadmium binding capacity of Zn-induced hepatic MT in vitro. To examine the possible protective effect of Zn independent of induction of MT synthesis, CCl4 was administered 2 h following Zn pretreatment and the hepatotoxic response was examined 4 h later. This study revealed limited protection by Zn prior to the induction of MT synthesis. These data further support a role of MT in the modulation of CCl4 hepatotoxicity.

Altered sex differentiation of hepatic ethylmorphine N-demethylation in the male rat following neonatal methadone exposure.

Since acute methadone treatment reduces serum androgen levels in adult male rats, we have investigated the effect of exposure of neonatal rats to methadone (M) on the "neonatal imprinting" of hepatic monooxygenase by testosterone. The kinetic constants for ethylmorphine N-demethylation (ED) in hepatic microsomes of adult rats are sex-related (female Km greater than male Km and male Vmax greater than female Vmax). Treatment of male neonates with M (2.5 mg/kg, sc) during the first 5 postnatal days resulted in an elevation of the Km, but no significant changes in the Vmax for ED in adult life. The M-induced "feminization" of the Km for ED could be prevented by a single dose of testosterone propionate (TP) given to the neonates on day 2 following birth. M treatment also reduced in a reversible manner neonatal serum androgen levels: Decreased androgen levels were not observed beyond the fifth postnatal day. These data suggest that the feminizing effect of M may be related to a reversible chemical castration effect during a critical neonatal period. Our studies also show that the imprinting of the Km and Vmax for ED do not occur during the identical period.

Metabolic activation/deactivation reactions during perinatal development.

The role of metabolic activation/deactivation reactions during development is evaluated in relation to developmental pharmacology and toxicology. Enzyme systems evaluated include the mixed-function oxidases (aryl hydrocarbon hydroxylase and oxidative demethylation), epoxide hydration and conjugation (glutathione conjugation, sulfation, and glucuronidation). Placental transfer and milk secretion of chemicals are discussed in relation to maternal, placental, and fetal metabolism. Normal patterns of enzyme development can be modified in two ways: (1) enzyme induction and (2) enzyme imprinting. Postnatal induction of the mixed-function oxidases and glucuronyl-transferase following treatment of pregnant rats with TCDD is shown to be caused primarily by newborn exposure to TCDD in milk. Structure-activity relationship are defined for the perinatal induction of hepatic enzymes by the pure PCBs. PCBs are divided into two classes: P-450 inducers and P-448 inducers. Imprinting or programming of hepatic metabolism is a function of the sexual differentiation of enzyme activity; male and female activities are similar in prepubertal animals, whereas pronounced sex differences are evident in adults. Treatment of newborn rats (days 2--6) with diethystilbestrol or testosterone resulted in a feminization (decrease) of mixed-function oxidation and glucuronidation in adult males. No changes were seen in immature males or females or adult females. This effect appears to be irreversible and is under pituitary-hypothalamic-gonadal control. In addition to the feminization of enzyme activity, neonatal exposure to hormonally active chemicals also feminizes the hepatic response to cadmium in resultant adult animals.

Functional modification of indole binding site with indomethacin congeners.

Indomethacin and four congeners were shown by equilibrium dialysis to interfere with the binding of L-tryptophan at the primary indole binding site on defatted human serum albumin. Incubation of albumin with 1-(p-bromoacetamidobenzovl)- and 1-(m-bromoacetamidobenzoyl)-5-methoxy-2-methyl-3-indolylacetic acid functionally modified this site, decreasing the availability of the site for the binding of L-tryptophan. Incubation in the presence of a large excess of L-tryptophan resulted in the partial protection of the site from modification by 1-(m-bromoacetamidobenzoyl)-5-methoxy-2-methyl-3-indolylacetic acid.

Dependence of gel-filtered guinea pig hepatic microsomes on a soluble factor for maximum mono-oxygenase activities.

Guinea pig hepatic microsomes were prepared by gel filtration on Sepharose 2B. A second, peak containing protein and hemoproteins (Hp) markedly increased mono-oxygenase activities when added to the microsomes. Gel filtration of guinea pig microsomes prepared by differential centrifugation revealed possible dissociation during elution from the Sepharose column. Parallel experiments with rat liver were negative.