D. Tsang

Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain

The ontogenesis of opiate receptors in several brain regions of the developing rat was measured by specific binding of [3H]methionine-enkephalin ([3H]Met). The amount of [3H]Met bound varied significantly with brain region as well as with age: a transient peak was observed at day 4 post-partum (p.p.) in the cerebellum; a hump was observed at days 8-17 in the brain stem; while in the whole forebrain the adult level was reached at day 17 p.p. Administration of morphine or naloxone to female rats before and during pregnancy significantly altered the regional development of [3H]Met binding sites in the brains of the offspring. These data suggest that the development of [3H]Met binding sites in brain follows a caudal to rostral sequence and that antenatal exposure to opiates affects the development of opiate receptors in brain. The latter effect may be responsible for the neonatal withdrawal symptoms in infants born to addicted mothers.

Requirement of PPARα in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation

The peroxisome proliferator-activated receptor α (PPARα) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARα-null (KO) mice and their wild-type (WT) counterparts were investigated. Our results indicated that mice deficient in PPARα displayed hepatomegaly and hypoketonemia following 72 h starvation. Histochemical analyses revealed that severe fatty infiltration was observed in the livers of KO mice under fasted conditions. Furthermore, 72 h fasting resulted in a 2.8-fold higher accumulation of hepatic TG in KO mice than in WT mice fasted for the same length of time. Surprisingly, the total hepatic PL contents in fasted KO mice decreased by 45%, but no significant change in hepatic PL content was observed in WT mice following starvation. Gas chromatographic analysis indicated that KO mice were deprived of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids during fasting. Taken together, these results show that PPARα plays an important role in regulation of fatty acid metabolism as well as phospholipid homeostasis during energy deprivation.

Baicalein inhibits DMBA-DNA adduct formation by modulating CYP1A1 and CYP1B1 activities

Flavonoids are phenolic compounds isolated from plants, and several of them like genistein and quercetin, have been documented to be effective in preventing cancer. Baicalein, a flavonoid extracted from the root of Scutellaria species, is widely used as a health supplement and herbal medicine in Asian countries. In this study, the chemopreventive effect of baicalein on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage was evaluated in an established cell culture model. In a preliminary screening, baicalein was identified to be a strong inhibitor to EROD activities induced by DMBA in MCF-7 cells. Subsequent enzyme kinetic analysis revealed that baicalein was a competitive inhibitor to EROD, and CYP1A1 and CYP1B1 gene expressions were also determined. Baicalein could reduce the CYP1A1/1B1 mRNA expressions induced by DMBA, and the mRNA abundance of CYP1A1 appeared to be more responsive than that of CYP1B1. A XRE-luciferase gene reporter assay indicated that AhR transactivation was suppressed. Since CYP1A1/1B1 were responsible for the biotransformation of polycyclic aromatic hydrocarbons, baicalein also demonstrated its ability to reduce DMBA-DNA adduct formation in MCF-7 cells. This study suggested that the natural occurring baicalein could be an agent preventing carcinogen-DNA adduct formation.

Effects of tumor necrosis factor alpha on taurine uptake in cultured rat astrocytes.

Taurine is known to play a major role in volume regulation in astrocytic swelling associated with stroke and brain trauma. Apart from brain edema, the severity of brain injury is related to the levels of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). TNFalpha had been shown to be closely associated with brain edema formation since the neutralization of TNFalpha reduced brain edema. Considering taurine has osmoregulatory functions in astrocytes, experiments were performed to study the effects of TNFalpha on taurine uptake in cultured astrocytes. Astrocytes exposed to 20 ng/ml of TNFalpha for 48 h showed a 91% increase in taurine uptake and significant increase was observed after 24 h exposure. This cytokine caused neither significant changes in cell volume nor taurine release. The increased in taurine uptake induced by TNFalpha was unlikely resulted from the modification of Na(+) movement because TNFalpha decreased tyrosine uptake, Na(+)-dependent transport system. In contrast to TNFalpha, interferon-gamma (IFNgamma) did not significantly affect taurine uptake. Taken together, our results did not support a suggestion that TNFalpha affects cell volume regulation via modulating taurine uptake in astrocytes. Increasing lines of evidence have demonstrated that taurine has anti-inflammatory and anti-oxidative effects, these findings therefore suggested that the increase in taurine uptake might be an adaptive response or a tool for astrocytes against oxidative stress.

Postnatal changes in [3H]mazindol-labelled dopamine uptake sites in the rat striatum following prenatal cocaine exposure

The present study showed that prenatal cocaine exposure (60 mg/kg/day) has a transient effect on the [3H]mazindol-labelled dopamine uptake sites in the striatum of the rat offspring examined from postnatal week 0-32. There is a 39% and 21% decrease in the number of binding sites (Bmax) in the cocaine-exposed group at postnatal weeks 3 and 4, respectively, with a recovery to near normal values by postnatal week 8.

Induction of tumor necrosis factor receptor type 2 gene expression by tumor necrosis factor-α in rat primary astrocytes

Using reverse transcription-polymerase chain reaction (RT-PCR) technique, the messenger RNA (mRNA) for tumor necrosis factor receptor type 2 (TNF-R2, 75/80 kDa) was detected in rat primary astrocytes, with much lower level of expression when compared to that for tumor necrosis factor receptor type 1 (TNF-R1, 55/60 kDa). Upon exposure to TNF-alpha (100 U/ml), the TNF-R2 mRNA level was greatly enhanced at 8 h, while TNF-R1 mRNA remained unchanged even after 24 h. The induction of TNF-R2 gene expression by TNF-alpha was dose-dependent and seemed to be unique to TNF-alpha, as interleukin-6 (IL-6) had no significant effect on TNF-R2 expression. Since TNF-R2 was reported to mediate mitogenic and gene-inducing effects in many other cell types, it is likely that the reported proliferative effect of TNF-alpha on astrocytes was also mediated by this TNF receptor subtype. Upon exposure to TNF-alpha or lipopolysaccharide (LPS), the expression of TNF-alpha gene was induced, and the LPS-induced TNF-alpha seemed to selectively enhance the TNF-R2 gene expression. Collectively, our results suggest that the TNF-alpha or LPS-induced expression of both TNF-R2 and TNF-alpha may provide a positive control mechanism to further enhance the proliferative effect of TNF-alpha in astrocytes.

Axonal sprouting in the hemisected adult rat spinal cord

The morphological and biochemical changes were studied in adult Sprague-Dawley rats after hemisection at the L3 spinal cord level. After survival periods of one, two and three months, fluorescent tracers, FluoroGold or rhodamine B, were implanted into the dorsal white columns of these rats at the positions of the corticospinal tract below the lesion. Following uptake of the tracer, the rats were killed and the motor cortices and spinal cords of both control and hemisected rats were analysed for positively labelled neurons. The highest number of labelled cells were found two months after hemisection. They were present in both sides of the cortices, particularly in the contralateral cortex, and also in the gray matter of the spinal cord above the hemisection. A few rats which were subjected to complete transection of the spinal cord also showed labelling of neurons in the motor cortex two months after lesion. The Protargol silver technique and the [3H]choline uptake study confirmed the presence of nerve fibres traversing the lesion site in the hemisected spinal cord. Furthermore, when the rats that had been hemisected two months earlier were subjected to a second cut at the same site, chromatolytic neurons were observed in the spinal cord as well as in the motor cortices of both sides. The hemisected rats demonstrated limited recovery in limb movement. The evidence of this study clearly shows that sprouting of nerve fibres has occurred in the lesioned adult rat spinal cord.

Screening of chemopreventive tea polyphenols against PAH genotoxicity in breast cancer cells by a XRE-luciferase reporter construct.

Polycyclic aromatic hydrocarbons (PAHs) are established cancer initiators that can be found in our food and environment. Some dietary plant polyphenols are strong inhibitors to PAH-induced mutagenesis, whereas others may not be as effective. To identify the chemopreventive compounds from a huge volume of dietary components, the development of an efficient screening method is required. In this study, a xenobiotic response element (XRE)-luciferase reporter plasmid was constructed to screen for some potential chemopreventive agents in tea against PAH-induced DNA damage. Tea is one of the most consumed beverages worldwide, and its beneficial effects on health have been documented. Previous studies have claimed that tea polyphenols could be protective against various cancers, and the rich database can be a source for comparison. Among the green and black tea polyphenols, the XRE-luciferase reporter assays suggested that only epigallocatechin gallate (EGCG) was effective in reducing XRE-driven luciferase assay in MCF-7 cells at the concentrations tested. Further study indicated EGCG could reduce CYP1A1 and CYP1B1 mRNA abundances and decrease the DMBA-DNA lesions. The results of DNA covalent binding of all tea polyphenols tested were consistent with the XRE-reporter assays. This study illustrated that the XRE-reporter assay was a viable screening test for dietary chemopreventive agents against PAH-initiated breast mutagenesis. It has the advantages of shorter sample processing time and producing no radioactive waste over directly measuring the CYP1A1/1B1 expressions, DNA lesion, or gel mobility shift assay.

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.

Tumor necrosis factor-α mediates the proliferation of rat C6 glioma cells via β-adrenergic receptors

In the present study, we observed that isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, stimulated rat C6 glioma cell proliferation, while propranolol, a beta-AR blocker, greatly reduced the proliferative effect of TNF-alpha on C6 cells. The gene and protein expressions of both beta1- and beta2-ARs were enhanced in C6 cells after TNF-alpha treatment, and the increase in beta-AR was due to an increased number of binding sites and not due to increase in receptor affinity. We further showed that protein kinase C (PKC) was involved in the TNF-alpha-induced beta-AR expression. Collectively, our results indicate that TNF-alpha-induced proliferation in C6 glioma cells might be via the induction and activation of beta-ARs.