Antioxidant, oxidative DNA damage protective and antimicrobial activities of the plant Trigonella foenum-graecum.

BACKGROUND: The plant Trigonella foenum-gracecum (TFG) is used as antidiabetic and diuretic. In order to ascertain antioxidant potential of leaf (early and mature) and seed of TFG, total phenolics, free radical scavenging assay, superoxide anion radical scavenging activity, reducing power, lipid peroxidation, ferric thiocyanate assay, hydroxyl radical scavenging activity and DNA damage protective activities were determined. The study was further carried out to assay the antimicrobial activity and HPLC analysis of plant parts. RESULTS: Ethanol extracts of leaf (early and mature) exhibited a high content of phenolics (54.79 and 41.28 g kg(-1) GAE) when it was compared with seed extract (23.85 g kg(-1) GAE). Results showed that mature TFG leaf extract had the lowest IC50 for the free radical scavenging assay (IC50 = 2.23 mg mL(-1)), superoxide anion radical scavenging activity (IC50 = 2.71 mg mL(-1)), hydroxyl radical scavenging activity (IC50 = 17.30 mg mL(-1)) and highest reducing power (10.14 ascorbic acid equivalents mL(-1)). However, the ethanol seed extract showed the maximum inhibition of lipid peroxidation and the ferric thiocyanate assay. Mature leaf also showed the maximum DNA damage protection activity and higher concentration of phytochemicals. CONCLUSION: The results showed that the mature TFG leaf had a higher antioxidant activity, which may be due to the presence of total phenolics. It may be used in herbal drugs or as a nutritional supplement.

Interaction of drug metabolizing cytochrome P450 2D6 poor metabolizers with cytochrome P450 2C9 and 2C19 genotypes modify the susceptibility to head and neck cancer and treatment response.

The present case-control study attempted to investigate the association of poor metabolizer (PM) genotypes of cytochrome P450 2D6 (CYP2D6*4 and CYP2D6*10) with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving chemotherapy or combination of chemo- and radiotherapy. Cases with the PM genotypes of CYP2D6 displayed a significantly increased risk for HNSCC as compared to wild type genotypes. The risk was found to further increase in cases (up to 4.8) carrying combination of PM genotypes of CYP2D6, CYP2C9 (CYP2C9*2) or CYP2C19 (CYP2C19*2), suggesting that synergism amongst the PM genotypes of drug metabolizing CYPs leads to impairment in the detoxification of the tobacco carcinogens. A small increase in the risk in tobacco (chewers or smokers) or alcohol users in cases with CYP2D6*4 allele while no change or even a small decrease in risk in cases with CYP2D6*10 allele when compared to non-tobacco or alcohol users have suggested that CYP2D6 genotypes alone do not appear to interact significantly with environmental risk factors in modifying the susceptibility to HNSCC. Furthermore, most of the cases carrying PM genotypes of CYP2D6 did not respond to the treatment. Moreover, higher prevalence of non-responders among cases carrying combination of CYP2D6*4 or CYP2D6*4, CYP2C9*2 and CYP2C19*2 have demonstrated that interaction of PM genotypes may not only significantly modify the susceptibility to HNSCC but also the treatment response.

Association of poor metabolizers of cytochrome P450 2C19 with head and neck cancer and poor treatment response.

A case-control study consisting of 300 patients and an equal number of healthy controls was carried out to investigate the association of polymorphism in cytochrome P450 2C19 (CYP2C19), which results in poor and extensive metabolizers (PMs and EMs) genotypes, with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving combination of chemo-radiotherapy. A higher frequency of CYP2C19 2 variants was observed in the cases resulting in significantly higher risk to HNSCC (Ad OR 3.36, 95% CI 1.94-5.82, p-value<0.05). The PM genotype of CYP2C19 3 was also found to be slightly increased in the cases, though the increase in risk was not significant when analyzed by multivariate logistic regression model. Tobacco chewing amongst the cases resulted in almost 13-fold increase in the risk with CYP2C19 2 (OR: 12.39) and 3-fold with CYP2C19 3 genotype (OR: 2.90) when compared to the tobacco chewers amongst the controls. Likewise, cigarette smoking in the cases increased the risk approximately 9-fold and 3-fold with CYP2C19 2 (OR: 8.93) and CYP2C19 3 (OR: 2.18) genotypes respectively when compared to smokers amongst the controls. Similar increase in risk was associated with alcohol use amongst the cases carrying variant genotypes of CYP2C19 2 (OR: 7.75) or CYP2C19 3 (OR: 2.60), demonstrating the importance of gene-environment interaction in modifying susceptibility to HNSCC. Interestingly, patients with PMs of CYP2C19 (CYP2C19 2 and CYP2C19 3) exhibited little response to the respective chemotherapy than the patients carrying wild-type genotype demonstrating that functional enzyme deficiencies due to polymorphism in CYPs may not only be important in modifying the susceptibility to HNSCC but also in determining chemotherapeutic response.

Rat testicular germ cell type(s) targeted by anti-spermatogenic agents in vivo and their recovery on withdrawal of treatment--a flow cytometric study.

Spermatogenesis goes through very critically and precisely balanced ratios of germ cells with diverse DNA ploidies (1C, 2C and 4C). Antispermatogenic agents that reversibly interrupt spermatogenesis may have a contraceptive relevance. With a view to study the precise mechanism of action of antispermatogenic agents and identify the germ cell type(s) targeted by various agents in vivo, spermatogenic cells with diverse DNA ploidies were measured in rat testis during treatment and recovery with compounds CDRI-84/35, gossypol and estradiol, using Flow Cytometry. Rats were treated with either CDRI-84/35 (100mg/(kg day) for 15 days followed by 25mg/(kg day) for 55 days) or gossypol (20mg/(kg day) for 70 days) or estradiol benzoate (2.5microg/(rat day) for 70 days) and 3 rats from each group were sacrificed after 22, 41, 53 and 70 days of treatment to monitor the changes in population of 1C, 2C, S-phase and 4C germ cell types. Treatment with CDRI-84/35 resulted in a significant and rapid drop in 1C population with a concomitant and parallel rise in 2C population. In gossypol-treated animals 1C peak disappeared gradually and the arrest was seen predominantly at 2C stage and partially at 4C stage. At the end of the treatment most of the germ cells were arrested at 2C stage. Estradiol affected spermatogenesis differently with 1C population falling in complement to rise in both 2C and 4C peaks. Germ cells were mainly arrested at the 4C stage after the treatment. The data suggest that germ cells fail to enter meiosis in CDRI-84/35-treated rats. Few cells entering meiosis do not complete the cell division and remain arrested at 4C stage. However in case of estradiol and gossypol the meiotic 4C cells become incapable of further differentiation into haploid cells. After receiving 70 days of treatment a few rats were allowed to recover for 60, 90 and 120 days. The population of various germ cell types in the testis of recovery-group animals indicated that spermatogenesis resumes substantially in case of estradiol treatment and partially in case of treatment with the other two agents.

Long lasting effects of prenatal exposure to deltamethrin on cerebral and hepatic cytochrome P450s and behavioral activity in rat offspring.

Prenatal exposure to different doses (0.25, or 0.5 or 1.0 mg/kg corresponding to 1/320 th or 1/160 th or 1/80 th of LD50) of deltamethrin to the pregnant Wistar rats from gestation day 5 to 21 were found to produce a dose dependent increase in the activity of cytochrome P450 (CYP) dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-D) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of cytochrome P450 monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have indicated that increase in cytochrome P450 activity may lead to the accumulation of deltamethrin and its metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic cytochrome P450s was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.5 and 1.0 mg/kg) of deltamethrin and up to 9 weeks at the highest dose (1.0 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6 weeks of age, have further indicated that due to the reduced activity of the cytochrome P450s during the ontogeny, the pyrethroid or its metabolites accumulating in the brain may not be cleared from the brain, thereby leading to the persistence in the increase in the expression of cerebral and hepatic cytochrome P450s in the offspring postnatally up to 9 weeks. The data suggests that low dose prenatal exposure to pyrethroids has the potential to produce long lasting effects on the expression of xenobiotic metabolizing cytochrome P450s in brain and liver of the offspring.

Peripheral blood leucocytes ornithine decarboxylase activity in chronic myeloid leukemia patients: prognostic and therapeutic implications.

Leukocytes ornithine decarboxylase (ODC) activity was measured in normal individuals and in patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP) as well as in accelerated phase (CML-AP), with an aim to examine the role of ODC activity in prognostic evaluation of CML patients. Our results showed that ODC activity was significantly higher in CML-CP (41.02+/-25.57nmol/h per 10(7) cells, P<0.005) and CML-AP (67.71+/-44.42nmol/h per 10(7) cells, P<0.001) patients than in normal subjects (3.12+/-1.34nmol/h per 10(7) cells). Furthermore, patients with CML-AP showed higher ODC activity than CML-CP patients (P<0.005). Patients with CML-CP who converted to accelerated phase within 24 months had higher ODC activity (84.58+/-12.81nmol/h per 10(7) cells) than patients who did not convert to accelerated phase (31.13+/-18.24nmol/h per 10(7) cells). The high value of ODC activity was also associated with less clinico-hematological response. We suggest that ODC activity reflects the neoplastic proliferative activity in CML patients and may serve as an additional prognostic marker.

Probiotic Enterococcus lactis IITRHR1 protects against acetaminophen-induced hepatotoxicity.

OBJECTIVE: Acetaminophen (APAP), an antipyretic/analgesic drug, is reported to cause toxicity on overdose. Dietary supplements are currently being explored to decrease toxicity. In the present study, the protective effect of probiotic Enterococcus lactis IITRHR1 was evaluated at different doses (10(7), 10(8), and 10(9) colony-forming units) against APAP-induced liver damage. METHODS: Male Wistar rats were administered APAP (1 g/kg of body weight orally) for 14 d, and hepatotoxicity was assessed by marker enzymes in serum and observation of histopathologic changes. Rats were pretreated with probiotic E. lactis IITRHR1 for 7 d and modulation of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase), redox ratio, and ferric reducing antioxidant power was assessed. Oxidative damage by APAP to membrane lipids, proteins, and DNA was also observed. Involvement of Bax, Bcl2, cytochrome c (pro-/anti-apoptotic proteins), caspases, and their modulation was assessed by immunoblot analysis and reverse transcriptase polymerase chain reaction. RESULTS: The E. lactis IITRHR1 pretreatment lowered the level of biomarkers of hepatotoxicity in serum. A significant increase was observed in the level of antioxidant enzymes and redox ratio and decreased oxidative damage to membrane lipids and proteins. Probiotic E. lactis IITRHR1 also modulated key apoptotic/anti-apoptotic proteins such as cytochrome-c, Bcl2, Bax, expression of caspases, and resultant DNA damage. CONCLUSION: Probiotic strain E. lactis IITRHR1 was found to have antioxidant capacity and afforded protection against APAP-induced hepatotoxicity by modulating antioxidant status, pro-/anti-apoptotic proteins, caspases, and DNA damage.

Cytochrome P4503A: evidence for mRNA expression and catalytic activity in rat brain.

Studies initiated to investigate the presence of cytochrome P4503A (CYP3A) isoenzymes in brain revealed constitutive mRNA and protein expression of CYP3A1 in rat brain. Western blotting studies showed that pretreatment with CYP3A inducer such as pregnenolone-16alpha -carbonitrile (PCN) significantly increased the cross reactivity comigrating with hepatic CYP3A1 and CYP3A2 in rat brain microsomes. RT-PCR studies have also shown increase in mRNA expression of CYP3A1 following pretreatment of rats with PCN. The ability of rat brain microsomes to catalyze the demethylation of erythromycin, known to be mediated by CYP3A isoenzymes in liver and significant increase in the activity of erythromycin demethylase (EMD) following pretreatment with dexamethasone or PCN have indicated that CYP3A isoenzymes expressed in brain are functionally active. Kinetic studies revealed that increase in the enzyme activity following pretreatment with PCN resulted in increase in the apparent affinity (Km) and Vmax of the reaction. Similarities in the inhibition of the constitutive and inducible brain and liver EMD activity following in vitro addition of ketoconazole, a inhibitor specific for CYP3A catalysed reactions and anti-CYP3A have further indicated that like in liver, CYP3A isoenzymes catalyse the activity of EMD in rat brain. Data also revealed regional differences in the activity of EMD in the brain. Relatively higher constitutive as well as inducible mRNA expression of CYP3A1 in hypothalamus and hippocampus, the brain regions responsive to steroid hormones have suggested that CYP3A isoenzymes may not only be involved in the process of detoxication mechanism but also in the metabolism of endogenous substrates in brain.

Expression of constitutive and inducible cytochrome P450 2E1 in rat brain.

Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitro studies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.

Antioxidant status during cutaneous wound healing in immunocompromised rats.

The present investigation was undertaken to investigate the endogenous status of free radical scavengers during cutaneous wound healing in immunocompromised rats. Antioxidant contents and lipid peroxidation product in terms of malondialdehyde (MDA) have been monitored in the wound tissues of immunosuppressed rats at different time intervals (2, 7 and 14 days) following cutaneous injury. A significant increase in MDA content and decrease in glutathione and vitamin C content was observed in the skin of immunocompromised rats as compared to control subjects. Further, a significant decrease in vitamin C, vitamin E content, catalase and glutathione peroxidase activity was observed at 2 days postwounding in immunocompromised rats. A significant and time-dependent decrease in glutathione content was also observed at 7 and 14 days postwounding. However, the healing tissue on 2 and 7 days postwounding exhibited significantly elevated superoxide dismutase activity. The MDA content was augmented only at 2 days postwounding in immunosuppressed rats. Thus significant alterations in the antioxidant profile accompanied by elevated levels of MDA, a marker of free radical damage may be contributory to impaired wound healing in immunocompromised rats.

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids.

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.