Serrine S. Lau

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration

While studying Bim, a BH3-only proapoptotic protein, we identified an ∼36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The ∼36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS–PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca2+ homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.

The role of metabolism in 3,4-(+)-methylenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity.

Abstract: 3,4-Methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are ring-substituted amphetamine derivatives with stimulant and hallucinogenic properties. The recreational use of these amphetamines, especially MDMA, is prevalent despite warnings of irreversible damage to the central nervous system. MDA and MDMA are primarily serotonergic neurotoxicants. Because (1) neither MDA nor MDMA produces neurotoxicity when injected directly into brain, (2) intracerebroventricular (ICV) administration of some major metabolites of MDA and MDMA fails to reproduce their neurotoxicity, (3) &agr;-methyldopamine (&agr;-MeDA) and N-methyl-&agr;-MeDA are metabolites of both MDA and MDMA, (4) &agr;-MeDA and N-methyl-&agr;-MeDA are readily oxidized to the corresponding ortho-quinones, which can undergo conjugation with glutathione (GSH), and (5) quinone thioethers exhibit a variety of toxicologic activities, we initiated studies on the potential role of thioether metabolites of &agr;-MeDA and N-methyl-&agr;-MeDA in the neurotoxicity of MDA and MDMA. Our studies have revealed that the thioether conjugates stimulate the acute release of serotonin, dopamine, and norepinephrine and produce a behavioral response commensurate with the “serotonin syndrome.” Direct injection of the conjugates into rat brain also produces long-term depletions in serotonin (5-HT) concentrations, elevations in GFAP expression, and activation of microglial cells. The data are consistent with the view that thioether metabolites of &agr;-MeDA and N-methyl-&agr;-MeDA contribute to the neurotoxicity of the parent amphetamines.

2,5-bis-(Glutathion-S-yl)-α-methyldopamine, a putative metabolite of (±)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations

3,4-(+/-)-Methylenedioxyamphetamine (MDA) and 3,4-(+/-)-methylenedioxymethamphetamine (MDMA) are serotonergic neurotoxicants. However, when injected directly into brain, MDA and MDMA are not neurotoxic, suggesting that systemic metabolism plays an important role in the development of neurotoxicity. The nature of the metabolite(s) responsible for MDA- and MDMA-mediated neurotoxicity is unclear. alpha-Methyldopamine is a major metabolite of MDA and is readily oxidized to the o-quinone, followed by conjugation with glutathione (GSH). Because the conjugation of quinones with GSH frequently results in preservation or enhancement of biological (re)activity, we have been investigating the role of quinone-thioethers in the acute and long-term neurochemical changes observed after administration of MDA. Although intracerebroventricular (i.c.v.) administration of 5-(glutathion-S-yl)-alpha-methyldopamine (4 x 720 nmol) and 5-(N-acetylcystein-S-yl)-alpha-methyldopamine (1 x 7 nmol) to Sprague-Dawley rats produced overt behavioral changes similar to those seen following administration of MDA (93 mumol/kg, s.c.) they did not produce long-term decreases in brain serotonin (5-hydroxytryptamine, 5-HT) concentrations. In contrast, 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine (4 x 475 nmol) decreased 5-HT levels by 24%, 65% and 30% in the striatum, hippocampus and cortex, respectively, 7 days after injection. The relative sensitivity of the striatum, hippocampus and cortex to 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine was the same as that observed for MDA; the absolute effects were greater with MDA. The effects of 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine were also selective for serotonergic nerve terminal fields, in that 5-HT levels were unaffected in regions of the cell bodies. Because 2,5-bis-(glutathion-S-yl)-alpha-methyldopamine caused long-term depletion in 5-HT without adversely affecting the dopaminergic system, it also mimics the selectivity of MDA/MDMA. The data imply a possible role for quinone-thioethers in the neurobehavioral and neurotoxicological effects of MDA/MDMA.

Reactive intermediates and their toxicological significance

Many chemicals that cause toxicity do so via metabolism to biologically reactive metabolites. However, the nature of the interaction between such reactive metabolites and various cellular components, and the mechanism(s) by which these interactions eventually lead to cell death are poorly understood. The relative importance of macromolecular alkylation (covalent binding), lipid peroxidation, alterations in thiol, calcium and energy homeostasis are discussed with reference to specific toxicants. It is concluded that the cytotoxic effects of reactive metabolites are a consequence of simultaneous and/or sequential alterations in several cellular processes. Further studies are required to determine the relationship between these alterations and cell death.

The Cytoprotective Effect of N-acetyl-L-cysteine against ROS-Induced Cytotoxicity Is Independent of Its Ability to Enhance Glutathione Synthesis

2,3,5-Tris(glutathion-S-yl)-hydroquinone (TGHQ), a metabolite of hydroquinone, is toxic to renal proximal tubule epithelial cells. TGHQ retains the ability to redox cycle and create an oxidative stress. To assist in elucidating the contribution of reactive oxygen species (ROS) to TGHQ-induced toxicity, we determined whether the antioxidant, N-acetyl-L-cysteine (NAC), could protect human kidney proximal tubule epithelial cells (HK-2 cell line) against TGHQ-induced toxicity. NAC provided remarkable protection against TGHQ-induced toxicity to HK-2 cells. NAC almost completely inhibited TGHQ-induced cell death, mitochondrial membrane potential collapse, as well as ROS production. NAC also attenuated TGHQ-induced DNA damage and the subsequent activation of poly (ADP-ribose) polymerase and ATP depletion. Moreover, NAC significantly attenuated c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase phosphorylation induced by TGHQ. In contrast, NAC itself markedly increased extracellular regulated kinase1/2 (ERK1/2) activation, and the upstream mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, PD-98059, only partially inhibited this activation, suggesting that NAC can directly activate ERK1/2 activity. However, although NAC is frequently utilized as a glutathione (GSH) precursor, the cytoprotection afforded by NAC in HK-2 cells was not a consequence of increased GSH levels. We speculate that NAC exerts its protective effect in part by directly scavenging ROS and in part via ERK1/2 activation.

DNA adducts, genetic polymorphisms, and K-ras mutation in human pancreatic cancer

To test the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis in susceptible individuals, aromatic DNA adducts and 8-hydroxyguanosine (8-OH-dG) were measured by (32)P-postlabeling and HPLC-EC, respectively, in 31 pancreatic tumors and 13 normal tissues adjacent to the tumor from patients with pancreatic cancer. Normal pancreatic tissues from 24 organ donors, from six patients with non-pancreatic cancers, and from five patients with chronic pancreatitis served as controls. It was found that tissue samples from patients with pancreatic cancer had significantly higher levels of both aromatic DNA adducts and 8-OH-dG compared with control samples. The mean (+/-S.D.) levels of aromatic DNA adducts were 101.8+/-74.6, 26.9+/-26.6, and 11.2+/-6.6 per 10(9) nucleotides in adjacent tissues, tumors, and controls, respectively. The mean (+/-S.D.) levels of 8-OH-dG were 11.9+/-9.6, 10.8+/-10.6, and 6.7+/-4.6 per 10(5) nucleotides in adjacent tissues, tumors, and controls, respectively. Polymorphisms of the CYP1A1, CYP2E1, NAT1, NAT2, GSTM1, MnSOD, and hOGG1 genes were determined in these patients. The level of aromatic DNA adducts was significantly associated with polymorphism of the CYP1A1 gene. No significant correlation was found between the level of 8-OH-dG and the MnSOD, GSTM1, and hOGG1 polymorphisms. However, one novel polymorphism/mutation of the hOGG1 gene was found in a pancreatic tumor. Mutation at codon 12 of the K-ras gene was found in 25 (81%) of 31 pancreatic tumors, including three G-to-A transitions and 22 G-to-T transversions. Patients with the G-to-T mutation had a significantly higher level of aromatic DNA adducts than those with G-to-A or wild-type codon (P=0.02). On the other hand, the K-ras mutation profile was not related to the level of 8-OH-dG. Given the limitation of sample size, these preliminary data lend further support the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis.

The contribution of bromobenzene to our current understanding of chemically-induced toxicities

The metabolism and toxicity of bromobenzene has been investigated for well over one hundred years. The urinary excretion of mercapturic acids was first reported in 1879, in animals treated with bromobenzene. Bromobenzene has since proven to be a valuable tool in efforts to unravel the complexities involved in chemical- induced toxicities. For example, the importance of metabolic activation via the cytochrome(s) P-450; the role of glutathione in the detoxification of reactive metabolites; and the toxicological significance of covalent binding, enzyme inactivation and lipid peroxidation have all been illustrated in studies with bromobenzene. Thus, many of the principles involved in chemical-induced toxicity have been exemplified in studies with bromobenzene. These studies have provided substantial insight into the role of chemically reactive metabolites in the genesis of xenobiotic-mediated cytotoxicity.

Hepatic microsomal epoxidation of bromobenzene to phenols and its toxicological implication.

Abstract In vitro microsomal hepatic epoxidation of bromobenzene in rats and mice is presented in this study. Formation of o-bromophenol via bromobenzene-2,3-epoxide and p-bromophenol via bromobenzene-3,4-epoxide was assayed enzymatically and identified by a new, rapid and sensitive gas-liquid chromatography method using electron capture detection. Pretreatment of the animals with phenobarbital caused significant increases in both pathways whereas 3-methylcholanthrene or β-naphthoflavone caused a selective and marked increase of only the 2,3-epoxide pathway. Sodium dodecyl sulfate-gel electrophoresis of microsomal preparations resolved multiple forms of cytochrome P-450 and indicated that different forms of the heme protein were responsible for the formation of o-bromophenol and p-bromophenol. it is of interest that various inducers augment particular pathways for a common substrate especially since bromobenzene-3,4-epoxide and not the bromobenzene-2,3-epoxide has been proposed as the cytotoxic reactive metabolite of bromobenzene.

Improved MALDI-TOF imaging yields increased protein signals at high molecular mass.

Matrix assisted laser desorption ionization (MALDI) mass spectrum images are created from an array of mass spectra collected over a tissue surface. We have increased the mass range of proteins that can be detected in tissue sections from kidneys, heart, lung and brain of different rodent species by a modification of the sandwich technique, which involves co-crystallizing matrix with analyte. A tissue section is placed upon a drop of sinapinic acid matrix dissolved in 90% ethanol and 0.5% Triton X-100. Once the matrix has dried, a seed layer of sinapinic crystals is added as a dispersion in xylene. Additional layers of sinapinic acid are added as solutions in 90% ethanol followed by 50% acetonitrile. Numerous peaks with signal to noise ratio of four or greater are observed between 25 kDa to 50 kDa. This represents ∼10 times as many peaks as are detected using traditional matrix spotting and spraying.

The role of ortho-bromophenol in the nephrotoxicity of bromobenzene in rats

ortho-Bromophenol (1.92 mmol/kg, ip) caused a 50% decrease in renal glutathione levels within 90 min. In contrast, hepatic glutathione levels remained 80% of control values 5 hr after ortho-bromophenol administration. Renal glutathione was far more susceptible to the initial rapid depleting effects of ortho-bromophenol than was hepatic glutathione, the dose-response curve for hepatic glutathione depletion being shifted to the right. ortho-Bromophenol at doses greater than 1.6 mmol/kg caused severe renal necrosis in noninduced rats, with consequent elevations in BUN levels. This dose was one-fifth as large as that required by bromobenzene to produce a similar necrosis in phenobarbital-treated rats (W. D. Reid, Exp. Mol. Pathol., 19, 197-214, 1973). Phenobarbital pretreatment and depletion of tissue glutathione with diethyl maleate caused significant increases in BUN levels over controls. Pretreatment with piperonyl butoxide decreased the incidence of elevated BUN levels following ortho-bromophenol administration. While liver microsomes converted ortho-bromophenol to covalently bound material, kidney microsomes did not. However, in vivo, ortho-bromophenol covalently bound to kidney protein of control rats four times greater than to liver protein. Phenobarbital pretreatment increased the in vivo covalent binding to kidney protein but not to liver protein. The degree of covalent binding to kidney protein correlated with BUN levels (r = 0.91, p less than 0.001). The nature of the nephrotoxic metabolite of ortho-bromophenol is not known, but an intermediate may be generated in the liver and transported to the kidney. These findings suggest that ortho-bromophenol may play a role in the nephrotoxicity observed following bromobenzene administration.