Antidyslipidemic and antioxidant activity of an unusual amino acid (2-amino-5-hydroxyhexanoic acid) isolated from the seeds of Crotalaria juncea.

In continuation of our drug discovery programme on Indian medicinal plants, we isolated an unusual amino acid, i.e. 2-amino-5-hydroxyhexanoic acid (1) from the seeds of Crotalaria juncea. The 2-amino-5-hydroxyhexanoic acid (1) showed dose dependent lipid lowering activity in the in vivo experiments and also showed good in vitro antioxidant activity. The cyclized compound, 3-amino-6-methyltetrahydro-2H-pyran-2-one (2) showed better lipid lowering and antioxidant profile than the parent compound 1.

Antidyslipidemic and antioxidant activity of the constituents isolated from the leaves of Calophyllum inophyllum.

In continuation of our drug discovery program on Indian medicinal plants, we isolated bioactive compounds (1-5) from the leaves of Calophyllum inophyllum and evaluated their antidyslipidemic activity in triton induced hyperlipidemia model. The calophyllic acid (1A) and isocalophyllic acid (1B) mixture, canophyllic acid (4) and amentoflavone (5) showed dose dependent lipid lowering activity in in vivo experiments. The compounds 1A+1B mixture and 3 also showed good in vitro antioxidant activity.

Effect of chromium on the level of IL-12 and IFN-gamma in occupationally exposed workers.

Chromium may affect humoral and cellular immunity, acting on T lymphocytes as well as on granulocytes and monocytes cells. Cytokines play an important role in the immune balance. In this study, the level of IL-12 and IFN-gamma were evaluated in the sera and PHA/LPS stimulated culture supernatant of human PBMCs of healthy volunteers and occupationally exposed chromium workers. All the workers were highly exposed to chromium having mean of 104.65+/-77.21 microg/dL (range 23.7-316.8 microg/dL). A suspension of exposed and unexposed human PBMC (0.5x10(6) cells/ml) prepared and cultured in RPMI-1640 supplemented with 10% FCS for 18 h in the presence or absence of LPS (10 ng/ml) which used for stimulation of IL-12 and IFN-gamma. The level of IL-12 and IFN-gamma were evaluated in the sera samples as well as LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers. In these chromium exposed workers the level of IL-12 was 433.66+/-197.49 pg/ml and 983.45+/-330.99 pg/ml in LPS stimulated culture supernatant of normal individuals and highly chromium exposed workers, which was significant (P<0.05). Although the level of IL-12 was (78.61+/-61.03 pg/ml to 146.52+/-46.37 pg/ml) elevated in unstimulated culture supernatant of h-PBMCs of chromium exposed individuals as compared to control, but it was not significant. This observation also suggests that a significant increase in IFN-gamma production in LPS stimulated and unstimulated culture supernatant of h-PBMCs of chromium exposed workers as compared to control. However, IFN-gamma level have a significant positive correlation between blood chromium level (r=0.833, t=6.3872, P 0.05) and exposure time (in years) (r=0.8916, t=8.3540, P 0.05) of the occupationally exposed workers.

Symptom-specific care-seeking behavior for sick neonates among urban poor in Lucknow, Northern India.

To assess symptom-specific care-seeking practices for newborns and behavioral factors associated with them to inform strategies to enhance newborn care seeking in urban Lucknow, Northern India. This was a prospective follow-up study of consecutive 326 neonates delivered at an urban reproductive and child health (RCH) center. Focused Group Discussions (n=5) were also conducted in urban slums (n=3) at the RCH center (n=1) and at a district hospital (n=1). Overall, 326 neonates were recruited within 48 h of birth and 289 (88.7%) were followed up at 6 weeks (+/-15 days) at home. Parents of 51.2% (148/289) neonates reported at least one symptom of illness. Among these, 27.3% (79/289) neonates had at least one reported Integrated Management of Neonatal and Childhood Illnesses (IMNCI) danger sign, of which 15 (18.9%) did not receive any modern medical care, 5(33.3%) of which were dead by early infancy. Care seeking from unqualified providers (spiritual/traditional) was 33.3% (3/9) for persistent diarrhea and 23.5% (4/17) for pneumonia. Qualitative data from Focused Group Discussions showed that when pictures of some danger signs were shown like sunken eyes, reduced skin turgor, chest in-drawing and bulged fontanel, care seeking for these as well as fast breathing were influenced by 'local beliefs', which considered them to be untreatable by modern medicines alone. Thus, care seeking from multiple providers and use of traditional/home remedies delayed appropriate and timely medical care seeking. Almost half of the neonates had an illness symptom of which half had an IMNCI danger sign, of which one fifth did not receive medical care. Therefore, there is an urgent need to introduce a locally modified community IMNCI program here, for promoting care seeking from qualified providers for sick neonates.

Suppression of IL-6 level in human peripheral blood mononuclear cells stimulated with PHA/LPS after occupational exposure to chromium.

The toxic metals alter the immune response of animals as well as humans. In addition to the well documented and numerous toxic effect of chromium on various target organs, number of studies shown that acute and chronic exposure to inorganic chromium may result in impairment of immune functions in the experimental systems. Immunosuppression appears to be more subtle effect of exposure to heavy metals. Therefore, we have taken two different groups of chromium exposed individuals. These were leather tanning workers and chromeplaters. These groups of individuals were regularly exposed to chromium. All the leather tanning workers were highly exposed to chromium having a mean of 96.60+/-113.95 mg/dl (range 12.4-417.21 mg/dl). A suspension of exposed and unexposed human PBMC (0.5x10(6) cells/ml) prepared and cultured in RPMI-1640 supplemented with 10% FCS for 18 h in the presence or absence of PHA (5 microg/ml) and LPS (10 ng/ml) which used for stimulation of IL-2, IL-4, TNF-alpha, IL-10 and IL-6, respectively. The levels of Th1/Th2 cytokine: IL-2, IL-4, TNF-alpha, IL-10 and IL-6 were evaluated in the sera and PHA/LPS stimulated culture supernatant of human PBMCs of chromium exposed workers. In these workers the level of IL-6 was 543.95+/-123.75 pg/ml and 388.40+/-61.24 pg/ml in PHA/LPS stimulated culture supernatant of normal individuals and highly chromium exposed workers, which was significant (P<0.05). This observation suggests that IL-6 levels were suppressed in chromium exposed groups as compared to unexposed healthy volunteers. Although the level of IL-2 in PHA stimulated culture supernatant of PBMCs was suppressed in chromium exposed individuals but it was not significant, IL-4 and IL-10 could not be detected. However, there was no difference in TNF-alpha levels in sera samples as well as unstimulated culture supernatant of h-PBMCs of chromium exposed individuals as compared to control.

Use of benzimidazoles in children younger than 24 months for the treatment of soil-transmitted helminthiasis.

Considerable experience and limited quantitative evidence indicate that infections with the soil-transmitted helminths Ascaris lumbricoides and Trichuris trichiura usually start to become established in children aged 12 months and older. Since children living in countries where the infections are endemic are at risk of morbidity, even those as young as 12 months may need to be considered for inclusion in public health programmes designed to reduce morbidity by means of regular anthelminthic chemotherapy. This situation raises the question as to whether such young children should be given anthelminthic drugs. Systems for the absorption, distribution, metabolism and elimination of drugs do not fully develop until children are in their second year of life. Current knowledge, however, reveals that the incidence of side effects linked to benzimidazole drugs in young children is likely to be the same as in older children. Accordingly, we conclude that albendazole and mebendazole may be used to treat children as young as 12 months if local circumstances show that relief from ascariasis and trichuriasis is justified.

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation.

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.

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.

Effectiveness and cost-effectiveness of albendazole in improving nutritional status of pre-school children in urban slums.

OBJECTIVE: To study the clinical efficacy and the incremental cost-effectiveness of albendazole in improving the nutritional status of pre-school children. DESIGN: Single blind, placebo-controlled trial with child as the unit of randomization. SETTING: In the Anganwadi centers of the Integrated Child Development Services situated in the urban slums of Lucknow, North India. METHODS: Thirty-two Anganwadi centers were randomly selected for the trial. Included were registered resident children between 1.5 to 3.5 years of age with informed and written parental consent. The intervention group received 600 mg of albendazole powder every six months while the placebo group received same quantity of calcium powder. Enrolled children were contacted once in six months from January 1995 to 1997 and given treatment. The outcome measure were change in the proportion of underweight (weight for age <-2.00z), stunted (height for age <-2.00z) children and the cost per child prevented from becoming stunted. RESULTS: There were 610 and 451 children in the albendazole and placebo groups, respectively. Mean age at recruitment was 31.8 months (SD: 9.7). Follow-up and compliance in both the groups was >95%. During the 2 year follow-up, the proportion of stunted children increased by 11.44% and 2.06% in the placebo and albendazole groups, respectively, and the difference was 9.38% (95% CI 6.01% to 12.75%; p value <0.0001). Direct fecal smear was positive for the ova of ascaris in 41.2% and 55.3% children in the albendazole and placebo groups, respectively at the end of the study (p value <0.001). The annual family expenditure on illness in the recruited child was Rs. 743 (SD: 662) and Rs. 625 (SD: 609) in the albendazole and the placebo groups, respectively. The incremental cost-effectiveness ratio was Rs 543.00 for each case of stunting prevented with albendazole. There was no difference in the various morbidity or cognitive performance, as judged by the revised Denver prescreening questionnaire, in both the groups at enrollment as well as at the end of the study. CONCLUSIONS: Six monthly albendazole reduces the risk of stunting with a small increase in the expenditure on health care from the payer's perspective. Larger trials are needed to study the effect of albendazole on prevention of stunting, cognitive functions and all-cause childhood mortality.

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.

Early prediction of neonatal hyperbilirubinemia.

The study aim was to predict, using serum bilirubin level measured 18 to 24 hours (SB, 18-24) after birth, the occurrence of peak serum bilirubin level > 15 mg/dL (hyperbilirubinemia) or the requirement of phototherapy, any time from the second to fifth postnatal day. The study was conducted on a prospective cohort of 274 neonates born in north India. The main outcome measures were (a) hyperbilirubinemia and (b) phototherapy. Serum bilirubin level was estimated at 18-24 hours of age and then daily from second to fifth postnatal day. Exclusion criteria were Rh incompatibility, asphyxia and life threatening congenital malformations; and neonates of women with gestational diabetes or history intake of drugs affecting the fetal liver. Hyperbilirubinemia was found in 12.8%; and 19.3% neonates received phototherapy. Dichotomous SB 18-24, using a cut-off of > 3.99 mg/dL, as the "prediction test" had the following sensitivity and specificity for predicting (a) hyperbilirubinemia: 67% and 67%, respectively, and (b) the treatment with phototherapy: 64% and 68%, respectively. We concluded that by using SB 18-24 as the "prediction test", approximately two-thirds of neonates were test negative and had about one in ten chances of re-admission for treatment of hyperbilirubinemia, if discharged. After further validation, our results will be of benefit to neonates delivered in developing countries.

Induction of glutathione S-transferase pi as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model.

There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase pi (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)-anti-7,8-dihydroxy-9, 10-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [(+)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP-induced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r = -0.89; p < 0.05) as well as in the forestomach (r = -0.97; p < 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model.

Oxidative changes in brain of aniline-exposed rats.

Oxidative stress in rat cerebellum, cortex and brain stem after a short-term high-dose exposure to aniline vapors under conditions akin to those after major chemical accidents, was studied. Significant increases in superoxide dismutase isozyme activities and formation of thiobarbituric acid reactive material along with depletion of ascorbic acid and non-protein sulfhydryl content suggest impairment of antioxidant defenses 24 h after single exposure to 15,302 ppm aniline vapors for 10 min.