Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: Removal of O-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 μmol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O⁶-methylguanine (O⁶-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P < 0.05), and colonic (at three sequential time points; ANOVA, F = 4.96, P < 0.05) O⁶-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P < 0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P = 0.0026, r = 0.83, r² = 0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

Vanadium induces apoptosis and modulates the expressions of metallothionein, Ki-67 nuclear antigen, and p53 during 2-acetylaminofluorene-induced rat liver preneoplasia

Our previous studies have shown that vanadium, a dietary micronutrient, has an inhibitory effect against experimentally induced rat hepatocarcinogenesis. In this study, we evaluated the role of vanadium on some potential protein expression markers of carcinogenesis, such as metallothionein (MT), an intracellular metal-binding protein linked with cell proliferation and apoptosis, Ki-67 nuclear antigen, and p53 tumor suppressor during 2-acetylaminofluorene (2-AAF)-induced (0.05% in basal diet) rat liver preneoplasia. In a short-term regimen, supplementation of vanadium at a dose of 0.5 ppm effectively suppressed the formation of DNA 'comets' (29.55%; P < 0.02), thereby indicating its nongenotoxicity at this particular dose. Vanadium administration throughout the study reduced relative liver weight (RLW), nodular incidence (57.15%), total number, and multiplicity (48.45%) with restoration of hepatic zinc (Zn), magnesium (Mg), selenium (Se), copper (Cu), iron (Fe), and calcium (Ca) contents when compared to the carcinogen control. Moreover, treatment with vanadium significantly abated the expressions of MT and Ki-67, studied at four sequential time points. An increased immunopositivity of p53 protein (1.03 +/- 0.23%; P < 0.02) was found in vanadium-treated rat liver with an elevated apoptotic-labeling index (AI; P < 0.001) as documented by TUNEL assay. Furthermore, a positive correlation between MT expression and Ki-67 labeling along with a strong negative correlation between MT immunoreactivity and AI (r = -0.9000, P = 0.0004 at week 24) at various time intervals suggest that, vanadium-mediated suppression of MT and Ki-67 expressions may be associated with induction of apoptosis. The results thus provide evidence for the first time in support of the potential role of vanadium on induction of p53 and apoptosis with concurrent suppression of MT and Ki-67 in order to have an understanding, in part, of the chemopreventive mechanism of this trace element in limiting neoplastic transformation in a defined model of experimental rat hepatocarcinogenesis.     

Vanadium inhibits DNA-protein cross-links and ameliorates surface level changes of aberrant crypt foci during 1,2-dimethylhydrazine induced rat colon carcinogenesis

The trace mineral vanadium inhibits cancer development in a variety of experimental animal models. The present study was to gain insight into a putative anticancer effect of vanadium in a rat model of colon carcinogenesis. The in vivo study was intended to clarify the effect of vanadium on DNA-protein cross-links (DPC), surface level changes of aberrant crypt foci (ACF) and biotransformation status during 1,2-dimethylhydrazine (1,2-DMH) induced preneoplastic rat colon carcinogenesis. The comet assay showed statistically higher mean base values of DNA-protein mass (p<0.01) and mean frequencies of tailed cells (p<0.001) in the carcinogen-induced group after treatment with proteinase K. Treatment with vanadium in the form of ammonium monovanadate supplemented ad libitum in drinking water for the entire experimental period caused a significant (p<0.02) reduction (40%) in DNA-protein cross-links in colon cells. Further, the biotransformation status of vanadium was ascertained measuring the drug metabolising enzymes, glutathione S-transferase (GST) and cytochrome P-450 (Cyt P-450). Significantly, there was an increase in glutathione S-transferase and cytochrome P-450 levels (p<0.01 and p<0.02, respectively) in rats supplemented with vanadium as compared to their carcinogen controls. As an endpoint marker, we also evaluated the effect of vanadium on surface level changes of aberrant crypt foci induced by 1,2-DMH by scanning electron microscopy. Animals induced with 1,2-DMH and supplemented with vanadium showed a marked improvement in colonic architecture with less number of aberrant crypt foci in contrast to the animals induced with 1,2-DMH alone, thereby exhibiting its anticarcinogenicity by modulating the markers studied herein.     

Protective role of vanadium on the early process of rat mammary carcinogenesis by influencing expression of metallothionein, GGT-positive foci and DNA fragmentation

Vanadium, a dietary micronutrient, is now proving to be a promising anti-tumour agent. The present study was conducted to ascertain its anti-neoplastic potential against an experimental mammary carcinogenesis. Female Sprague-Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(alpha)anthracene (DMBA; 0.5 mg per 100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 p.p.m. was supplemented in the drinking water and given ad libitum to the experimental group immediately after the carcinogen treatment and it continued until the termination of the study (24 weeks for histological, immunological and biochemical observations and 35 weeks for morphological findings). It was found that vanadium treatment brought about substantial protection against DMBA-induced mammary carcinogenesis. This was evident from histological findings that showed substantial repair of hyperplastic lesions following supplementation of vanadium alone. There was a significant reduction in incidence (P<0.05), total number, multiplicity (P<0.01), size of palpable mammary tumours and delay in mean latency period of tumour appearance (P<0.001) following vanadium supplementation compared to the DMBA control. The immunohistochemical localization of metallothionein (a prognostic marker for breast cancer) showed reduced expression with vanadium treatment. Further, DNA fragmentation in the mammary tissue of the vanadium-treated group indicated apoptosis. In this group, vanadium also caused a significant decrease in the number (P<0.002) and focal area (P<0.05) of gamma-glutaminetranspeptidase-positive hepatic foci. The results clearly show the anti-neoplastic potential of vanadium.     

Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure,function, and stability

O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.     

Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides,a possible role in radioprotection

The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides (RH-3) which has already been reported to render more than 80% protection against radiation induced mortality in mice. Direct and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose degradation and 2,2-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion.RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 500 g/ml). 2,2-bipiridyl assay revealed the inability of RH-3 to chelate Fe²⁺ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks in a dose dependent manner and at concentrations of 100 and 120 g/ml the length of comet tail was considerably reduced and became almost similar to that of untreated control. RH-3 at a concentration of 120 g/ml or more induced a strong compaction of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resistant even to a radiation dose of 1000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5°C respectively; however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21°C correspondingly.RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts would be necessary.     

Influence of substituents at C11 on hydroxylation of progesterone analogs by Bacillus sp

Transformation of progesterone analogs viz., progesterone, 11 alpha-, 11 beta-hydroxyprogesterones and 11-ketoprogesterone by Bacillus sp. is reported. Both progesterone and 11-ketoprogesterone were hydroxylated while the C(11) epimeric alcohols of progesterone remained unaltered under the conditions used. The major bioconverted products obtained from progesterone and 11-ketoprogesterone were characterized as 6 beta- and 14 alpha-hydroxyprogesterones and 14 alpha-hydroxy-11-ketoprogesterone respectively by mass and NMR spectra. The conversion of 11-ketoprogesterone to its 14 alpha-hydroxy derivative by microbe is unprecedented and novel. Moreover, hydroxylation at 6 beta- and 14 alpha-positions of progesterone by Bacillus sp. is significant. In conclusion, the present data showed that the substituents at 11-position of steroid play important role on hydroxylation by microbe.     

QSAR study on the affinity of some arylpiperazines towards the 5-HT1A/alpha1-adrenergic receptor using the E-state index

QSAR models represent the relationship of biological activity with either physicochemical parameters or structural indices. QSAR study was performed on some arylpiperazines as 5-HT(1A)/alpha(1)-adrenergic receptor antagonists using E-state indices to identify the pharmacophoric requirements. It was found that some of the atoms played important roles to both activities and some played important role in selectivity of compound to the 5-HT(1A) antagonistic activity. The presence of COONHPr group at the ortho-position of the phenyl ring might be disadvantageous and Br at meta-position might be conducive to the activity. COOPr at the ortho-position might be disfavored the adrenergic alpha(1)-antagonistic activity, thus increase the selectivity.     

Geometry of interaction of the histidine ring with other planar and basic residues

Among the aromatic residues in protein structures, histidine (His) is unique, as it can exist in the neutral or positively charged form at the physiological pH. As such, it can interact with other aromatic residues as well as form hydrogen bonds with polar and charged (both negative and positive) residues. We have analyzed the geometry of interaction of His residues with nine other planar side chains containing aromatic (residues Phe, Tyr, Trp, and His), carboxylate (Asp and Glu), carboxamide (Asn and Gln) and guanidinium (Arg) groups in 432 polypeptide chains. With the exception of the aspartic (Asp) and glutamic (Glu) acid side-chains, all other residues prefer to interact in a face-to-face or offset-face-stacked orientation with the His ring. Such a geometry is different from the edge-to-face relative orientation normally associated with the aromatic-aromatic interaction. His-His pair prefers to interact in a face-to-face orientation; however, when both the residues bind the same metal ion, the interplanar angle is close to 90 degrees. The occurrence of different interactions (including the nonconventional N-H...pi and C-H...pi hydrogen bonds) have been correlated with the relative orientations between the interacting residues. Several structural motifs, mostly involved in binding metal ions, have been identified by considering the cases where His residues are in contact with four other planar moieties. About 10% of His residues used here are also found in sequence patterns in PROSITE database. There are examples of the amino end of the Lys side chain interacting with His residues in such a way that it is located on an arc around a ring nitrogen atom.