Utpal Pandya

Curcumin–glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.

Dietary curcumin prevents ocular toxicity of naphthalene in rats

Administration of naphthalene is known to cause cataract formation in rats and rabbits and naphthalene-initiated cataract is frequently used as a model for studies on senile cataract in humans. Oxidative stress has been implicated in the mechanism of naphthalene-induced cataract. Curcumin, a constituent of turmeric, a spice used in Indian curry dishes, is an effective antioxidant and is known to induce the enzymes of glutathione-linked detoxification pathways in rats. During the present studies, we have examined whether low levels of dietary curcumin could prevent naphthalene-induced opacification of rat lens. The presence of apoptotic cells in lens epithelial cells was also examined by catalytically incorporating labeled nucleotide to DNA with either Klenow fragment of DNA polymerase or by terminal deoxynucleotidyl transferase (TdT), which forms polymeric tail using the principle of TUNEL assay. The results of these studies demonstrated that the rats treated with naphthalene and kept on a diet supplemented with only 0.005% (w/w) curcumin had significantly less opacification of lenses as compared to that observed in rats treated only with naphthalene. Our studies also demonstrate, for the first time, that naphthalene-initiated cataract in lens is accompanied and perhaps preceded by apoptosis of lens epithelial cells and that curcumin attenuates this apoptotic effect of naphthalene.

The effect of curcumin on glutathione-linked enzymes in K562 human leukemia cells

Curcumin, an antioxidant present in the spice turmeric (Curcuma longa), has been shown to inhibit chemical carcinogenesis in animal models and has been shown to be an anti-inflammatory agent. While mechanisms of its biological activities are not understood, previous studies have shown that it modulates glutathione (GSH)-linked detoxification mechanisms in rats. In the present studies, we have examined the effects of curcumin on GSH-linked enzymes in K562 human leukemia cells. One micromolar curcumin in medium (16 h) did not cause any noticeable change in glutathione peroxidase (GPx), glutathione reductase, and glucose-6-phosphate dehydrogenase activities. Gamma-glutamyl-cysteinyl synthetase activity was induced 1.6-fold accompanied by a 1.2-fold increase in GSH levels. GSH S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene, and 4-hydroxynonenal (4HNE) were increased in curcumin-treated cells 1.3- and 1.6-fold, respectively (P = 0.05). The GST isozyme composition of K562 cells was determined as follows: 66% of GST Pl-1, 31% of Mu class GST(s), and 3% of an anionic Alpha-class isozyme hGST 5.8, which was immunologically similar to mouse GSTA4-4 and displayed substrate preference for 4HNE. The isozyme hGST 5.8 appeared to be preferentially induced by curcumin, as indicated by a relatively greater increase in activity toward 4HNE. Immunoprecipitation showed that GPx activity expressed by GST 5.8 contributed significantly (approximately 50%) to the total cytosolic GPx activity of K562 cells to lipid hydroperoxides. Taken together, these results suggest that GSTs play a major role in detoxification of lipid peroxidation products in K562 cells, and that these enzymes are modulated by curcumin.

Attenuation of galactose cataract by low levels of dietary curcumin

Abstract Our previous studies have shown that curcumin, a dietary antioxidant present in turmeric, attenuates 4-hydroxynonenal induced lens opacification in organ culture (Awasthi et al , Amer. J. Clin. Nutr. 1996;64:761–766). Oxidative stress has been implicated in the mechanism of galactosemic cataract formation. Therefore, the present studies were designed to examine whether dietary curcumin can attenuate galactosemic cataract in rats in-vivo . 8 week old Sprague-Dawley rats were randomized into four groups and fed with either AIN-76 diet or that containing 30% galactose with or without curcumin 0.0025% (ww) in the diet. Progression of cataract formation was monitored by slit lamp biomicroscopy at days 1, 22 and 29. The eyes were removed out at day 29 and the lenses were dissected out. Images of the isolated lenses were acquired using a digital imager. The lens epithelium was dissected and lens epithelial cells were examined for apoptosis. Lenses of the animals fed a diet containing neither galactose nor curcumin and those fed only a curcumin containing diet remained transparent. The transparency of lenses from rats without or with curcumin was identical as assessed by measuring the average intensity of transmitted light (AITL) (241 ± 4, n=12). Lenses from galactose fed animals without curcumin were partially opacified with an AITL value of 77 ± 9 % of the controls, (n=10, p

Global transcription profiling and virulence potential of Streptococcus pneumoniae after serial passage

Streptococcus pneumoniae, a facultative human pathogen associated with wide variety of diseases, such as pneumonia, meningitis, sepsis and otitis media. Factors involved in the initial colonization, survival and etiology of this commensal bacteria in the human host from the ex vivo environment is still not clearly understood. Here, we report alterations in global transcriptional profiles of S. pneumoniae 6304 serotype 4 after 50 passages (50P) and 100 passages (100P) on laboratory media to better understand gene expression strategies employed by the bacterium during progression from the nasopharynx to the blood. The results show that six-fold more genes were differentially expressed after 100P as compared to 50P. After 100P on blood agar plates, 726 genes (33%) of 2192 genes in the S. pneumoniae genome were differentially expressed. Moreover, the majority of these genes (68%) were expressed at higher levels with increasing passage number and from different functional groups. Significantly, all the genes present in Region of Diversity 10 (RD10) are required for virulence during blood stream infection showed enhanced expression after passage. However, there was no significant decrease in the LD(50) of serial passage strains compare to single passage strain in a mouse challenge model. Overall, our data suggest that bacteria adapt to extended laboratory passage by substantially altering gene expression. Furthermore, extended passage on blood agar plates reduces the expression of genes associated with initial colonization and adherence but enhances the expression of genes needed for systemic infection.