Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats

Chronic exposure to copper induces hepatocellular apoptosis with greater injury in the periportal region compared to the perivenous region. Here we have identified the factors responsible for the development of regional damage in the liver under in vivo conditions. Enhanced production of reactive oxygen species (ROS) with predominance of superoxide radical (O2•−) indicates the contribution of redox imbalance in the process. This may be linked with copper catalyzed oxidation of GSH to GSSG resulting in the generation of O2•−. Downregulation of Cu-Zn SOD in consequence of the degradation of this enzyme, causes decreased dismutation of O2•−, that further contributes to the enhanced level of O2•− in the periportal region. Decreased functioning of Mn SOD activity, reduction in mitochondrial thiol/disulphide ratio and generation of O2•− were much higher in the mitochondria from periportal region, which point to the involvement of this organelle in the regional hepatotoxicity observed during copper exposure. This was supported by copper-mediated enhanced mitochondrial dysfunction as evident from ATP depletion, collapse of mitochondrial membrane potential (MMP) and induction of mitochondrial permeability transition (MPT). Results suggest the active participation of O2•− in inducing mitochondrial dysfunction preferentially in the periportal region that eventually leads to the development of hepatotoxicity due to copper exposure under in vivo condition

Protective Effect of Andrographolide against Copper Mediated Hepatological Disorders in Rats.

Copper toxicosis with hepatic copper accumulation is associated with chronic cholestasis which eventually leads to cirrhosis of the liver. Liver is considered to be the main site for copper homeostasis in the body, and excess of this transition metal induces apoptotic changes in the hepatocytes followed by cirrhosis which eventually culminates in liver failure. We were interested to find out the underlying mechanism leading to the development of copper toxicity and to find out a short term treatment strategy against it. For this purpose, we have developed an animal model with rats. In the first chapter, we have established the fundamental mechanism of copper toxicity developed in liver of young rats. Chronic exposure to copper induces hepatocellular apoptosis with greater injury in the periportal region compared to the perivenous region. Here we have identified the factors responsible for the development of regional damage in the liver under in vivo conditions. Enhanced production of reactive oxygen species with predominance of superoxide radical indicates the contribution of redox imbalance in the process. This may be linked with copper catalyzed oxidation of reduced glutathione to oxidized glutathione resulting in the generation of superoxide radical. Downregulation of copper-zinc superoxide dismutase in consequence of the degradation of this enzyme, causes decreased dismutation of superoxide radical that further contributes to the enhanced level of superoxide radical in the periportal region. Decreased functioning of manganese superoxide dismutase activity, reduction in mitochondrial thiol/disulphide ratio and generation of superoxide radical were much higher in the mitochondria from periportal region, which point to the involvement of this 2 organelle in the regional hepatotoxicity observed during copper exposure. This was supported by copper-mediated enhanced mitochondrial dysfunction as evident from adenosine triphosphate depletion, collapse of mitochondrial membrane potential and induction of mitochondrial permeability transition. Results suggest the active participation of superoxide radical in inducing mitochondrial dysfunction preferentially in the periportal region that eventually leads to the development of hepatotoxicity due to copper exposure under in vivo condition. In the second chapter, we have ascertained a treatment schedule with andrographolide and D-penicillamine used in combination manner, which gives a promising result against copper induced liver damage in rats. Copper toxicity results from the cytoplasmic copper accumulation in periportal zone of liver due to intracanalicular cholestasis. We have shown that chronic exposure to copper caused oxidative stress and induced hepatocellular apoptosis in rats leading to periportal fibrosis. Despite several studies on the treatment of copper toxicosis, the result of these experimental therapies to date remains unsatisfactory. D-penicillamine, a metal chelator, still represents the drug of choice for the treatment of copper toxicosis. Long treatment regime and side effects after prolonged use remains a major hindrance to successful treatment with D-penicillamine. Effective short term treatment strategies involve coadministration of this agent with another hepatoprotective drug, andrographolide which is our drug of choice. Andrographolide exerts choleretic and anticholestatic effects and reverts back the cholestasis efficiently. Andrographolide potentiates the cytoprotective effect of D-penicillamine by accentuating the simultaneous removal of copper through bile and urine respectively. So, co-administration of andrographolide with D-penicillamine reduces hepatocyte death. This is accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and activation of caspase cascade. Toxic events 3 are consequense of oxidative stress and generation of proinflmmatory cytokines, we suggest that the antiapoptotic activity of combination therapy with andrographolide and D-penicillamine can be related to their ability to prevent the liver damage associated with copper toxicosis. To conclude, simultaneous treatment with andrographolide and Dpenicillamine synergistically exert antifibrotic effect and may therefore have a therapeutic implication for reducing copper overload as well as hepatic fibrosis associated with copper toxicosis. A change in the feeding pattern among Indian children is the first option to prevent the development Indian Childhood Cirrhosis. There should be a greater awareness among pediatricians about this disease to enable early diagnosis. Most of the patients are diagnosed at a very late stage of this disease and as a result commonly leads to death. We hope that, the research findings of our study will have a good implication in human society and can be used successfully for the treatment of human population, suffering from copper toxicity in future

Combination Therapy with Andrographolide and D-Penicillamine Enhanced Therapeutic Advantage over Monotherapy with D-Penicillamine in Attenuating Fibrogenic Response and Cell death in the Periportal zone of Liver in Rats during Copper Toxicosis

Long treatment regime with D-penicillamine is needed before it can exert clinically meaningful benefits in the treatment of copper toxicosis. The consequence of long-term D-penicillamine treatment is associated with numerous side effects. The limitations of D-penicillamine monotherapy prompted us to search for more effective treatment strategies that could decrease the duration of D-penicillamine therapy. The present study was designed to evaluate the therapeutic potential of D-penicillamine in combination with another hepatoprotective drug, andrographolide in treatment of copper toxicosis in rats. D-penicillamine treatment led to the excretion of copper through urine. Addition of andrographolide to D-penicillamine regime appeared to increase protection of liver by increasing the biliary excretion of copper and reduction in cholestatic injury. The early removal of the causative agent copper during combination treatment was the most effective therapeutic intervention that contributed to the early rectification of fibrosis in liver. Combination treatment reduced Kupffer cells accumulation and TNFα production in liver of copper exposed rats. In particular, andrographolide mediated the anti-inflammatory effect by inhibiting the cytokine production. However, another possible mechanism of cytoprotection of andrographolide was decreasing mitochondrial production of superoxide anions that resulted in better restoration of mitochondrial dysfunction during combination therapy than monotherapy. Furthermore, ROS inhibition by combination regimen resulted in significant decline in activation of caspase cascade. Inhibition of caspases attenuated apoptosis of hepatocytes, induced by chronic copper exposure. In summary, this study suggested that added benefit of combination treatment over use of either agent alone in alleviating the hepatotoxicity and fibrosis associated with copper toxicosis

SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1) cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1)/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation

Sumoylation is essential for Stra13-dependent growth inhibition.

<p>(A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B–C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean ±SD.</p

Stra13 is sumoylated.

<p>(A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three α-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. β-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxα, or PIASy as indicated. Lysates were immunoprecipitated with Myc- agarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS.</p

Mutation of sumoylation sites abrogates Stra13-mediated growth suppression.

<p>(A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B–C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean ±SD. (E) Stra13^−/− MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments.</p

Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization.

<p>(A) mRNA levels of cyclin D1, p21^Cip/WAF, cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean ±SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter.</p