Interaction of anthraquinones of Cassia occidentalis seeds with DNA and Glutathione.

Consumption of Cassia occidentalis (CO) seeds has been associated with the hepatomyoencephalopathy (HME) in children. Recently, we have characterized the toxic anthraquinones (AQs) such as Emodin, Rhein, Aloe-emodin, Chrysophanol and Physcion in CO seeds and detected these moieties in the bio fluids of CO poisoning cases. As AQs were detected in the serum of HME patients, their interaction with key biomolecules including protein, DNA and glutathione (GSH) is imperative. In this regard, we have previously reported the interaction of these AQs with serum albumin protein and their subsequent biological effects. However, the interaction of these AQs with DNA and GSH remained unexplored. In the present work, we have studied the binding of these AQs of CO seeds with DNA and GSH by fluorescence spectroscopy, UV-vis spectral analysis, molecular docking, and biochemical studies. Results indicated a higher binding affinity for Emodin (Ka = 3.854 × 104 L mol-1 S-1), Aloe-emodin (Ka = 0.961 × 104 L mol-1 S-1) and Rhein (Ka = 0.034 × 104 L mol-1 S-1) towards calf thymus DNA may be associated with their higher cytotoxicity. Alternatively, Physcion and Chrysophanol which showed less cytotoxicity in our earlier studies exhibited very low DNA binding. The binding pattern of all these AQs is consistent with the in-silico data. Absorption spectroscopy studies indicated the possible formation of GSH conjugate with Aloe-emodin and Physcion. Further biochemical measurement of GSH and GSSG (Glutathione disulfide) following incubation with AQs indicated that Aloe-emodin (28%) and Rhein (30%) oxidizes GSH to GSSG more as compared to other AQs. Taken together, these results suggest that the higher cytotoxicity of Rhein, Emodin and Aloe-emodin may be attributed to their potent DNA and GSH binding affinity.

Exosomes-based biomarker discovery for diagnosis and prognosis of prostate cancer.

African American men in the United States have higher incidence and mortality rates due to prostate cancer (PCa) compared to other races. In 2016 alone, nearly 30,000 cases of PCa in African American men were diagnosed and 4,450 men died from PCa. The underlying reasons for this health disparity in PCa are complex and include social, economic, and biologic determinants. To reduce or eliminate this health disparity, we must better understand the biology of the disease in African Americans and then develop novel diagnostic and prognostic biomarkers useful for timely and effective treatment decisions. Recently, there has been remarkable progress in understanding the role of exosomes (vesicles of 30-150 nm diameter) in cancer development and progression. Exosomes are loaded with unique cargo, including proteins, nucleic acids, lipids, and metabolites, that could predict the cells of their origin. Therefore, circulating exosomes in cancer patients are being used as a type of biopsy to identify novel biomarkers for early diagnosis, prognosis, and therapeutics. In this review, we discuss the promising use of exosomes to (a) identify race-related unique biological features of PCa, and (b) discover novel biomarkers for better diagnosis and prognosis of PCa, with the goal of reducing cancer health disparities.

Immunomodulatory potential of Rhein, an anthraquinone moiety of Cassia occidentalis seeds.

Rhein, the most toxic anthraquinone moiety in Cassia occidentalis seeds, has been associated with hepatomyoencephalopathy (HME) in children. Structural and functional alterations in the lymphoid organs have been reported both in HME patients and experimental animals indicating a possibility of the dysfunction of immune system following exposure to CO seeds or its toxic anthraquinones (Panigrahi et al., 2014a). In the present study the mechanism of immune response of Rhein in splenocytes has been investigated by measuring functional assays of lymphocyte, cell surface receptor expression and analysis of cytokine levels. Results indicate that Rhein at a maximum dose of 10 µM is non cytotoxic up to 72 h in splenocytes. In addition to its potential to decrease the allogenic response of T-cells, Rhein significantly suppresses the proliferation of the concavalin A (Con A) and lipopolysaccharide (LPS) stimulated splenocytes. Lymphocyte receptor expression analysis revealed that Rhein exposure significantly down regulate the expression of CD3e, CD4, CD8, CD28, CD69 molecules in T-cells. The expression of CD19, CD28, CD40 in B-cells were also found to be significantly decreased following Rhein exposure. In accordance with the functional responses, Rhein treatment significantly lowered the expression of IL2 and IL6 cytokines in Con A stimulated splenocytes, and IL6, IL10, IFNγ and TNFα in LPS stimulated splenocytes. Over all, the study suggests the immunomodulatory activity of Rhein and that it would be useful in understanding the immune response of CO seeds in human subjects.

Activity-guided chemo toxic profiling of Cassia occidentalis (CO) seeds: detection of toxic compounds in body fluids of CO-exposed patients and experimental rats.

Our prior studies have shown an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. However, the chemicals responsible for the CO poisoning are not known. Therefore, the present study was designed to identify the key moieties in CO seeds and their cytotoxicity in rat primary hepatocytes and HepG2 cells. Activity-guided sequential extraction and fractionation of the seeds followed by GC-MS analysis identified the toxic compounds in the CO seeds. These identified compounds were subsequently detected and quantified in blood and urine samples from CO-exposed rats and CO poisoning human study cases. GC-MS analysis of different fractions of methanol extracts of CO seeds revealed the presence of five anthraquinones (AQs), viz. physcion, emodin, rhein, aloe-emodin, and chrysophanol. Interestingly, these AQs were detected in serum and urine samples from the study cases and CO-exposed rats. Cytotoxicity analysis of the above AQs in rat primary hepatocytes and HepG2 cells revealed that rhein is the most toxic moiety, followed by emodin, aloe-emodin, physcion, and chrysophanol. These studies indicate that AQ aglycones are responsible for producing toxicity, which may be associated with symptoms of hepatomyoencephalopathy in CO poisoning cases.

Mechanism of rhein-induced apoptosis in rat primary hepatocytes: beneficial effect of cyclosporine A.

Past observational and toxicity studies have established an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. We recently reported chemical evidence of this association following the identification of toxic anthraquinones (AQs), viz. aloe-emodin, chrysophanol, emodin, physcion, and rhein, in CO seeds (Panigrahi, G. K. et al. (2015), Chem. Res. Toxicol. DOI: 10.1021/acs.chemrestox.5b00056 ). Of these five AQs, earlier studies have shown rhein to be the most cytotoxic AQ in hepatocytes. Therefore, the present study was designed to investigate the effect of rhein on rat primary hepatocytes. Results indicated that rhein (50 µM) causes apoptosis in rat primary hepatocytes by generating reactive oxygen species (ROS), increasing intracellular Ca(2+), decreasing the mitochondrial membrane potential, and depleting intracellular glutathione content. At the molecular level, rhein-induced DNA damage results in overexpression of γ-H2AX protein (2.5-fold), thereby causing enhancement of p53 (4.5-fold) and p21 (3.6-fold), leading to intrinsic pathway-mediated apoptosis involving Bax, bcl2, cytochrome c, caspases 3 and 9, and poly-ADP ribose polymerase. Further, it was observed that rhein-induced ROS generation is also involved in the modulation of signaling molecules like MAPK kinases, including ERK1/2, p38, and JNK, and mitochondrial energetics proteins, including complexes II-V, p-AMPK, and Sirt-1. It was shown that 100 nM cyclosporine A was the most effective among the different protective agents at preventing apoptosis in hepatocytes by interfering in various metabolic pathways which were found to be altered by rhein.

Hepatic transcriptional analysis in rats treated with Cassia occidentalis seed: involvement of oxidative stress and impairment in xenobiotic metabolism as a putative mechanism of toxicity.

The present study was undertaken to investigate the effect of Cassia occidentalis (CO) seeds on the transcriptional expression patterns of mRNAs in rat liver by microarray analysis. The results indicated that exposure of CO (0.5%) seeds in diet to rats differentially regulated 60 transcripts belonging to various metabolic pathways including, oxidative stress, xenobiotic metabolism, carbohydrate metabolism, cell cycle, apoptosis etc. The expression of AKT1, CAT, SOD1, CYP1A1, CYP2B1, TGF-ß, BAX, CREB1, JNK1 and IL-6 were validated by the qRT-PCR. In addition, involvement of oxidative stress was observed due to marked depletion of glutathione, increase in lipid peroxidation and modulation of antioxidant enzymes in hepatic tissue of rats treated with 0.5-2.0% CO in diet. Furthermore, significant decrease in the levels of Phase 1 (EROD, MROD and PROD) and Phase 2 (QR and GST) enzymes following 0.5-2.0% CO exposure indicates the impairment of xenobiotic metabolism and possible accumulation of toxic ingredients of the seeds in liver. Overall, the study predicts the involvement of multiple pathways and related biomolecules in CO induced hepatotoxicity and the data may be useful in formulating strategies for therapeutic interventions of suspected CO poisoning study cases.