Special Issue "Molecular Mechanisms of Organ-Specific Toxicity"

Abstract: Alzheimer’s disease (AD) is characterized by the abnormal aggregation of amyloid-β peptide (Aβ) in extracellular deposits known as senile plaques. The tyrosine residue (Tyr-10) is believed to be important in Aβ-induced neurotoxicity due to the formation of tyrosyl radicals. To reduce the likelihood of cross-linking, here we designed an Aβ-40 analogue (Aβ-40 Y10F) in which the tyrosine residue was substituted by a structurally similar residue, phenylalanine. The aggregation rate was determined by the Thioflavin T (ThT) assay, in which Aβ-40 Y10F populated an ensemble of folded conformations much quicker and stronger than the wild type Aβ. Biophysical tests subsequently confirmed the results of the ThT assay, suggesting the measured increase of β-aggregation may arise predominantly from enhancement of hydrophobicity upon substitution and thus the propensity of intrinsic β-sheet formation. Nevertheless, Aβ-40 Y10F exhibited remarkably decreased neurotoxicity compared to Aβ-40 which could be partly due to the reduced generation of hydrogen peroxide. These findings may lead to further understanding of the structural perturbation of Aβ to its fibrillation.

Abstract: Streptozotocin (STZ) is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.

Abstract: Compelling evidence has shown that exposure to anesthetics used in the clinic can cause neurodegeneration in the mammalian developing brain, but the basis of this is not clear. Neurotoxicity induced by exposure to anesthestics in early life involves neuroapoptosis and impairment of neurodevelopmental processes such as neurogenesis, synaptogenesis and immature glial development. These effects may subsequently contribute to behavior abnormalities in later life. In this paper, we reviewed the possible mechanisms of anesthetic-induced neurotoxicity based on new in vitro and in vivo findings. Also, we discussed ways to protect against anesthetic-induced neurotoxicity and their implications for exploring cellular and molecular mechanisms of neuroprotection. These findings help in improving our understanding of developmental neurotoxicology and in avoiding adverse neurological outcomes in anesthesia practice.

Abstract: Abstract: In this review, we elucidate the mechanisms of Aβ oligomer toxicity which may contribute to Alzheimer’s disease (AD). In particular, we discuss on the interaction of Aβ oligomers with the membrane through the process of adsorption and insertion. Such interaction gives rises to phase transitions in the sub-structures of the Aβ peptide from α-helical to β-sheet structure. By means of a coarse-grained model, we exhibit the tendency of β-sheet structures to aggregate, thus providing further insights to the process of membrane induced aggregation. We show that the aggregated oligomer causes membrane invagination, which is a precursor to the formation of pore structures and ion channels. Other pathological progressions to AD due to Aβ oligomers are also covered, such as their interaction with the membrane receptors, and their direct versus indirect effects on oxidative stress and intraneuronal accumulation. We further illustrate that the molecule curcumin is a potential Aβ toxicity inhibitor as a β-sheet breaker by having a high propensity to interact with certain Aβ residues without binding to them. The comprehensive understanding gained from these current researches on the various toxicity mechanisms show promises in the provision of better therapeutics and treatment strategies in the near future.

Abstract: Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg²⁺ has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2^−/− mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2^−/− mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells.

Abstract: Parkinson’s disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration. Evidence suggests that mitochondrial dysfunction may be linked to PD through a variety of different pathways, including free-radical generation and dysfunction of the mitochondrial Complex I activity. In Lewis rats, chronic systemic administration of a specific mitochondrial Complex I inhibitor, rotenone (3 mg/kg/day) produced parkinsonism-like symptoms. Increased oxidized proteins and peroxynitrite, and mitochondrial or cytosol translocation of Bim, Bax or cytochrome c in the striatum was observed after 2–4 weeks of rotenone infusion. After 28 days of systemic rotenone exposure, imunohistochemical staining for tyrosine hydroxylase indicated nigrostriatal dopaminergic neuronal cell degeneration. Characteristic histochemical (TUNEL or activated caspase-3 staining) or ultrastructural (electron microscopy) features of apoptotic cell death were present in the striatal neuronal cell after chronic rotenone intoxication. We conclude that chronic rotenone intoxication may enhance oxidative and nitrosative stress that induces mitochondrial dysfunction and ultrastructural damage, resulting in translocation of Bim and Bax from cytosol to mitochondria that contributes to apoptotic cell death in the striatum via cytochrome c/caspase-3 signaling cascade.

Abstract: Ursodeoxycholic acid (UDCA) is a steroid bile acid approved for primary biliary cirrhosis (PBC). UDCA is reported to have “hepato-protective properties”. Yet, UDCA has “unanticipated” toxicity, pronounced by more than double number of deaths, and eligibility for liver transplantation compared to the control group in 28 mg/kg/day in primary sclerosing cholangitis, necessitating trial halt in North America. UDCA is associated with increase in hepatocellular carcinoma in PBC especially when it fails to achieve biochemical response (10 and 15 years incidence of 9% and 20% respectively). “Unanticipated” UDCA toxicity includes hepatitis, pruritus, cholangitis, ascites, vanishing bile duct syndrome, liver cell failure, death, severe watery diarrhea, pneumonia, dysuria, immune-suppression, mutagenic effects and withdrawal syndrome upon sudden halt. UDCA inhibits DNA repair, co-enzyme A, cyclic AMP, p53, phagocytosis, and inhibits induction of nitric oxide synthatase. It is genotoxic, exerts aneugenic activity, and arrests apoptosis even after cellular phosphatidylserine externalization. UDCA toxicity is related to its interference with drug detoxification, being hydrophilic and anti-apoptotic, has a long half-life, has transcriptional mutational abilities, down-regulates cellular functions, has a very narrow difference between the recommended (13 mg/kg/day) and toxic dose (28 mg/kg/day), and it typically transforms into lithocholic acid that induces DNA strand breakage, it is uniquely co-mutagenic, and promotes cell transformation. UDCA beyond PBC is unjustified.

Abstract: Type 1 diabetes is characterized by insulin deficiency, type 2 by both insulin deficiency and insulin resistance: in both conditions, hyperglycaemia is accompanied by an increased cardiovascular risk, due to increased atherosclerotic plaque formation/instabilization and impaired collateral vessel formation. An important factor in these phenomena is the Vascular Endothelial Growth Factor (VEGF), a molecule produced also by Vascular Smooth Muscle Cells (VSMC). We aimed at evaluating the role of high glucose on VEGF-A₁₆₄ synthesis and secretion in VSMC from lean insulin-sensitive and obese insulin-resistant Zucker rats (LZR and OZR). In cultured aortic VSMC from LZR and OZR incubated for 24 h with D-glucose (5.5, 15 and 25 mM) or with the osmotic controls L-glucose and mannitol, we measured VEGF-A₁₆₄ synthesis (western, blotting) and secretion (western blotting and ELISA). We observed that: (i) D-glucose dose-dependently increases VEGF-A₁₆₄ synthesis and secretion in VSMC from LZR and OZR (n = 6, ANOVA p = 0.002–0.0001); (ii) all the effects of 15 and 25 mM D-glucose are attenuated in VSMC from OZR vs. LZR (p = 0.0001); (iii) L-glucose and mannitol reproduce the VEGF-A₁₆₄ modulation induced by D-glucose in VSMC from both LZR and OZR. Thus, glucose increases via an osmotic mechanism VEGF synthesis and secretion in VSMC, an effect attenuated in the presence of insulin resistance.

Abstract: The aim of this study was to determine if there are sex-related differences in the acute kidney injury induced by HgCl₂ since female rats express lower levels of renal Oat1 and Oat3 (transporters involved in renal uptake of mercury) as compared with males. Control males and females and Hg-treated male and female Wistar rats were employed. Animals were treated with HgCl₂ (4 mg/kg body weight (b.w.), intraperitoneal (i.p.)) 18 h before the experiments. HgCl₂ induced renal impairment both in male and female rats. However, female rats showed a lower renal impairment than male rats. The observed increase in kidney weight/body weight ratio seen in male and female rats following HgCl₂ treatment was less in the female rats. Urine volume and creatinine clearance decreased and Oat5 urinary excretion increased in both males and females, but to a lesser degree in the latter. Urinary alkaline phosphatase (AP) activity and histological parameters were modified in male but not in female rats after HgCl₂ administration. These results indicate that the lower Oat1 and Oat3 expression in the kidney of females restricts Hg uptake into renal cells protecting them from this metal toxicity. These gender differences in renal injury induced by mercury are striking and also indicate that Oat1 and Oat3 are among the main transporters responsible for HgCl₂-induced renal injury.

Abstract: Secreted clusterin (sCLU) has been shown to be overexpressed in metastatic hepatocellular carcinoma (HCC) tissue, and its overexpression in HCC cells increases cell migration and the formation of liver metastatic tumor nodules in vivo. In this study, we tested the hypothesis that sCLU plays a role in the invasiveness of human HCC and may be associated with its metastatic spread. HCCLM3, a human hepatocellular carcinoma cell line, was transiently transfected with an antisense oligonucleotide (ASO) against sCLU (OGX-011). HepG2 liver hepatocellular cells were transiently transfected with the pc.DNA3.1-sCLU plasmid to overexpress sCLU, and subsequently evaluated for effects on invasion and the expression of molecules involved in invasion. We observed that suppression of the sCLU gene significantly reduced the invasive capability of the highly invasive HCCLM3 cells, and vice versa in the low invasive HepG2 cell line. The results revealed that knockdown of sCLU by OGX-011 resulted in a significant increase in the expression of E-cadherin and a decrease in matrix metalloproteinase-2 (MMP-2) gene transcription. Overexpression of sCLU by transfection with pc.DNA3.1-sCLU significantly decreased the expression of E-cadherin and increased MMP-2 gene transcription. These data were further verified by reverse transcription-PCR and Western blot analysis. A significant reduction in MMP-2 expression and an increase in E-cadherin expression in sCLU-knockdown HCCLM3 cells were observed, as well as a significant increase in MMP-2 expression and a decrease in E-cadherin expression in HepG2 cells overexpressing sCLU. These data indicate a role for sCLU in augmenting MMP-2 transcription and decreasing E-cadherin expression. Our data show the involvement of sCLU in human HCC invasion, and demonstrate that silencing sCLU gene expression inhibits the invasion of human HCC cells by inhibiting MMP-2 expression and promoting E-cadherin expression. Thus, OGX-011 could be an effective therapeutic agent for HCC.

Abstract: The introduction of the so-called “targeted therapies”, particularly those drugs that inhibit the activity of tyrosine kinases, has represented a remarkable progress in the treatment of cancer. Although these drugs improve survival rates in cancer, significant cardiotoxicity, manifesting as left vertricular dysfunction and/or heart failure, has emerged. The ErbB receptor tyrosine kinases are being pursued as therapeutic targets because of their important roles in normal physiology and in cancer. Besides the fact that the ErbB receptors are indispensable during development and in normal adult physiology, epidermal growth factor (EGFR) and ErbB2 in particular have been implicated in the development of many human cancers. This review focuses on the rationale for targeting members of ErbB receptor family and numerous agents that are in use for inhibiting the pathway. We summarize the current knowledge on the physiological role of ErbB signaling in the ventricle and on structural aspects of ErbB receptor activation in cancer and cardiac cells. We examine the underlying mechanisms that result in on-target or off-target cardiotoxicities of ErbB inhibitors, which can influence the design of future anticancer therapies.

Abstract: Ricin toxin has been regarded as one of the most potent poisons in the plant kingdom, and there is no effective therapeutic countermeasure or licensed vaccine against it. Consequently, early detection of ricin intoxication is necessary. In this study, we took mice as test subjects, and used the technique of Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) and ClinProt™ microparticle beads to set up an effective detection model with an accuracy of almost 100%. Eighty-two peaks in the mass range 1000–10,000 m/z were detected by ClinProTools software, and five different peaks with m/z of 4982.49, 1333.25, 1537.86, 4285.05 and 2738.88 had the greatest contribution to the accuracy and sensitivity of this model. They may therefore provide biomarkers for ricin intoxication.