Pre-clinical toxicology and pathology of 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), a novel anti-cancer agent in male Beagle dogs.

We have developed a group of 4-substituted-1-nitroacridines with potent anti-tumor activity against prostate cancer and less toxic than parent 1-nitroacridines. The most active 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748) was selected for pre-clinical studies. The current study was undertaken to evaluate clinical and/or morphological adverse effects of C-1748 as a single intravenous dose at concentrations ranging from 0.16 to 4.6 mg/kg administered to male Beagle dogs. The maximum tolerated dose was 1.5 mg/kg. Emesis was observed in all groups lasting an average of 30 min to 12 h post-dosing. At high dose, extreme aggression was observed in one dog followed by disorientation and depression lasting for 48 h a frequent observation with chemotherapy. Reductions in platelets and white blood cells were observed which was similar to that seen with other chemotherapeutic agents. A compensatory hyperplasia of lymph nodes and a transient and limited extravasation in the intestinal mucosa were also observed. Increases in aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase were transient with normal levels restored by day 9. These enzyme increases were accompanied by epithelial hypertrophy of larger bile ductules in the periportal triads of the liver. The low toxicity profile and high tumor target activity make this novel class of drug a promising chemotherapeutic agent.

Pilot study using SELDI-TOF-MS based proteomic profile for the identification of diagnostic biomarkers of thyroid proliferative diseases.

Biomarkers for thyroid cancer (TCa) lack specificity. To develop TCa specific biomarkers, SELDI-TOF-MS was used to examine the proteomic profile of biopsies obtained from papillary TCa along with adjacent normal tissue. Sixty-three potential biomarkers were categorized by univariate analysis into single biomarker candidates and segregated by multivariate analysis into normal and cancerous groups. Our studies demonstrate the sensitivity and reproducibility of this approach to detect biomarkers for TCa.

Anti-carcinogenic and anti-metastatic properties of indole-3-carbinol in prostate cancer.

Indole-3-carbinol (I3C), a compound present as glucobracissin in cruciferous vegetables has anticancer activities which is in line with some of the epidemiological evidence that suggests a beneficial effect of consumption of cruciferous vegetables on cancer incidence and progression. The precise target of indole-3-carbinol has not been determined. We examined the effect of I3C on prostate cancer in a well-defined R3327 model using Copenhagen rats and the transplantable cell line, MAT-LyLu. This cell line derived from a tumor in Copenhagen rats is androgen independent and metastasizes to the lung and lymph nodes. Tumors were induced in Copenhagen rats by injecting MAT-LyLu subcutaneously and the animals treated with I3C that was administered either intraperitoneally or intravenously, in order to achieve maximal systemic exposure. This was a departure from the traditional chemopreventive route of indole-3-carbinol where the compound was incorporated in the diet. Our results indicate that I3C inhibited the incidence, growth and metastases of MAT-LyLu cells and both i.p. and i.v. injections of I3C were equally effective. Statistical analysis (Kaplan-Meier curves) clearly indicates a tumor-free and overall survival benefit as a result of treatment with I3C. These studies show for the first time that I3C in an injectible form has anti-prostate cancer activity.

Peptide mimotopes of oncoproteins as therapeutic agents in breast cancer.

Generation of an immune response to oncoproteins can lead to a cancer specific protective immunity. Several such oncoproteins are being examined as tumor targets with mixed results. We are evaluating the clinical utility of synthetic peptides that would mimic the antigen immunologically and elicit a tumor specific immune response. HER-2/neu, an oncoprotein whose expression in breast cancer is associated with poor prognosis, lower disease free-survival and a propensity for metastases was chosen as a model. Antibodies, Ab2, Ab4 and Ab5 directed towards the extracellular domain of HER-2/neu were reacted to peptides from two synthetic phage display peptide libraries, LX-8 (12-mer peptide library containing disulfide bridge) and X-15 (linear 15-mer). The isolated peptides were sequenced and characterized for ability to produce high titer antibodies and cross-reactivity. The peptides isolated did not show any sequence homology to protein databases but did show a hierarchy of immunogenic epitopes. Antibodies generated against peptides selected against the same antibody Ab2 or Ab4 showed affinity variation. Phages selected against Ab2 were also able to compete with binding of Ab2 to HER-2/neu. These results validate our hypothesis that synthetic peptides that mimic the antigenic epitope of oncoprotein can be generated and their clinical utility rests on devising a screening mechanism to identify peptides that can elicit an immune response directed to the oncoprotein and if possible its antigenic variants.

Induction of heat shock protein gp96 by immune cytokines.

Cytokines play a major role in regulating both humoral and cell-mediated immune responses. Recent advances in our understanding of cell-mediated immune responses have focused on the antigen presentation machinery and the proteins in the endoplasmic reticulum (ER). These proteins help the formation and stabilization of the major histocompatibility complex (MHC)-peptide interaction. A 96-kDa, ER-resident glycoprotein (gp96) is being evaluated as a therapeutic agent in cancer because of its ability to associate with a vast number of cellular peptides irrespective of size or sequence. Because the antigen presentation complex is assembled in the ER and a number of ER-resident proteins are modulated by cytokines, it is important to examine the regulation of gp96 in response to immune cytokines interferon gamma (IFN-gamma), and interleukin 2 (IL-2). Defects in signaling pathway in either of the cytokines can result in suboptimal immune response. We examined the effect of the cytokines IFN-gamma and IL-2 on the induction of gp96 in different cancer cell lines and examined the induction of DNA-binding proteins that recognize gamma interferon-activating sequence (GAS), present in the promoter region of gp96. The induction of GAS binding protein correlated with the induction of STAT 1 protein, a transcriptional regulator and mediator of IFN-gamma-mediated gene expression. The use of cytokines in inducing gp96 levels may have significance in maintaining high levels of gp96 for a sustained immune response.

Multiple molecular targets of indole-3-carbinol, a chemopreventive anti-estrogen in breast cancer.

The mechanism of action of the anti-estrogen indole-3-carbinol (I3C), present in cruciferous vegetables, is being examined in our laboratory with a view to promote the use of this naturally occurring chemopreventive as an alternative to synthetic anti-estrogens in human breast cancer. Our previous results clearly demonstrated that despite its low affinity for the estrogen receptor (ER), I3C abrogated estradiol-mediated cellular and biochemical effects in estradiol-responsive cells and tissues. In an earlier report, we identified ER phosphorylation as one of the targets of I3C, and in this communication we describe the consequence of inhibition of ER phosphorylation. Estradiol-induced DNA-binding proteins that bound to several DNA-responsive elements were inhibited by I3C and this effect was not at the level of DNA-protein physical interaction as inclusion of I3C in vitro in the reaction mix did not affect the binding. We analyzed the spectrum of genes induced by estradiol and modulated and/or intercepted by I3C. Our results conclude that although estradiol-mediated functions are affected by I3C, its biochemical targets are multiple and some of these may be modulated by the oligomeric products of I3C.

Mechanical, physico-chemical and microbial analysis of oil refinery waste receiving agricultural soil.

The mechanical, physico-chemical characteristics of soil and the activity of indigenous microflora were studied in the agricultural soil steadily receiving petroleum refinery effluent at Mathura, U.P, India The data on the soil grain size and texture revealed that the soil in the test region was basically loam or silty loam. Physic-chemical analysis showed considerable variability in the soil pH, temperature, moisture content and water holding capacity (WHC). A substantially higher microbial activity was noticed at the test sites as evident from the total variable (10(5) to 10(9) CFU g-1 soil) bacterial population. In addition, a significant population of proteolytic and cellulolytic bacteria, rhizobium and actinomycetes was detected. Oligotrophs were isolated and characterized into four types (I-IV). A fraction of oligotrophic bacteria, particularly those belonging to type II and type IV exhibited appreciable in distilled water. Invariably higher microbial biomass ranging from 366 to 1604 mg CO2. 100 g-1 soil, clearly implied that the soil in the test region was very well nourished and the refinery waste was providing enough nitrites to support the growth of soil microflora.

Proteasome inhibitors differentially affect heat shock protein response in cancer cells.

The heat shock proteins (HSPs) are molecular chaperones that are emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and intracellular targeting of peptides. The immunological function of the HSPs are imparted by tissue specific peptides associated with the HSPs and as such autologous cancer derived HSP-peptide complexes are unique therapeutic agents. Since a majority of the intracellular peptides are generated by the proteasome, we examined the consequence of abrogation of proteasome function by proteasome inhibitors (PIs) such as Lactacystin, MG-132 and LLM on the growth and induction profile of HSP70 and gp96 using hematopoietic, lymphoid, and epithelial derived cancer cell lines. The effect on growth was measured by the XTT assay and induction of the heat shock proteins by western blot analyses using HSP70 and gp96 specific antibodies. Of the PIs tested, cancer cells, were most sensitive to MG-132 and least sensitive to LLM. MG-132 also showed a 10-fold differential sensitivity between estrogen receptor positive, (ER+) MCF-7 cells and negative cells, (ER-) MDA-MB-231. Induction of heat shock proteins, gp96 and HSP70 was, however, noted in response to LLM. Since LLM exhibited minimal cytotoxic effect, metabolic stress that results in induction of HSPs may not be translated in cell growth inhibition and that there may exist a cell-type specific phenomenon in the HSP response to PI mediated metabolic stress.

Abrogation of estrogen-mediated cellular and biochemical effects by indole-3-carbinol.

The use of naturally occurring phytoantiestrogens for prevention and therapy of breast cancer is an alternative to synthetic antiestrogens. We have been examining the mechanism of action of the antiestrogen indole-3-carbinol (I3C), a constituent of compounds present in cruciferous vegetables. I3C abrogates the cell-proliferative effect of 17 beta-estradiol (E2), as observed in several different estradiol-responsive breast cancer cell lines and isolated cell clones. Modulation of E2 activity by I3C, in part, was by the induction of the 2-hydroxylation pathway, one of the two competing hydroxylation pathways of estrone conversion that resulted in the formation of metabolites with antiestrogenic properties. I3C-mediated induction of the 2-hydroxylation pathway correlated with a selective induction of cytochrome P-450 1A1 by I3C in E2-responsive human breast cancer cells. Induction of neither the 2-hydroxylation pathway nor cytochrome P-450 1A1 was observed in estrogen-nonresponsive human breast cancer cells. This selective effect warranted a further search for biochemical targets of I3C related to E2 function. To this end, we observed that E2-mediated phosphorylation of the estrogen receptor is inhibited by I3C. Our results are consistent with the hypothesis that I3C exerts its antiestrogenic effect by intervention in the E2-estrogen receptor signal transduction pathways and by alterations in E2 metabolism that resulted in the formation of metabolites with antiestrogenic activity.

The aging paradox: free radical theory of aging.

There are more than 300 theories to explain the aging phenomenon. Many of them originate from the study of changes that accumulate with time. Among all the theories, the free radical theory of aging, postulated first by Harman, is the most popular and widely tested, and is based on the chemical nature and ubiquitous presence of free radicals. This review aims to recapitulate various studies on the role of free radicals in DNA damage-both nuclear as well as mitochondrial-the oxidative stress they impose on cells, the role of antioxidants, the presence of autoantibodies, and their overall impact on the aging process.

Antigen binding characteristics of experimentally-induced antibodies against hydroxyl radical modified native DNA.

Hydroxyl radical, generated by UV irradiation of hydrogen peroxide caused damage to native calf thymus DNA leading to strand breaks, base modification and decrease in melting temperature. The ROS-DNA was highly immunogenic in goat. The antibody activity was inhibited to the extent of 89% with the immunogen as inhibitor. Antigenic specificity of anti-ROS-DNA antibodies by competition ELISA showed multiple cross-reactivity, recognizing B-, A- and allied conformations. The immune complex formation with native and ROS-DNA was substantiated by band shift assay. A striking observation, is the enhanced recognition of ROS-thymine and ROS-poly(dT) by the induced antibodies. These investigations suggest that ROS might be the plausible candidate for the presentation of unique epitopes on ROS-DNA, in particular of thymine, inducing antibodies cross-reacting with native DNA and play an important role in the pathogenesis of SLE.

Binding of human anti-DNA autoantibodies to reactive oxygen species modified-DNA and probing oxidative DNA damage in cancer using monoclonal antibody.

The binding of native and reactive oxygen species-modified DNA (ROS-DNA) to circulating antibodies in the serum of patients with various types of cancer has been investigated by competition enzyme-linked immunosorbent assay. Fifteen sera of 35 showed reactivity with native and/or ROS-DNA. Eleven of these showed higher binding to ROS-DNA (36-64% inhibition), whereas 1 showed higher reactivity with native DNA (nDNA) (42% inhibition). Three sera reacted with both native and ROS-DNA almost equally. Oxidative lesions in human genomic DNA were immunochemically detected using an anti-ROS-DNA monoclonal antibody (MAb) probe. Two of 3 DNA isolates from blood of breast cancer patients, 1 of 3 from lung cancer and 1 of 2 each from hepatocellular cancer and cancer of the gallbladder were reactive with the MAb. Higher recognition of ROS-DNA by circulating antibodies and DNA isolated from cancer patients by the MAb indicates increased oxidative stress leading to DNA damage. Our results suggest that ROS modification of DNA probably alters its immunogenicity leading to the generation of antibodies to ROS-DNA, probably by the activation of autoreactive cells. The induced antibodies against modified DNA are cross-reactive to native DNA.

Detection of oxidative DNA damage by a monoclonal antibody: role of lysyl residues in antigen binding.

Hydroxyl radical, a prominent entity of reactive oxygen species, is known to modify cellular DNA and has been implicated in several human diseases. A previously described monoclonal antibody (Mab) against reactive oxygen species-modified DNA (ROS-DNA), which preferentially recognizes ROS-modified epitopes on DNA, was used in this study. The epsilon-amino groups of lysine of the Mab were modified to study the role of these residues in Mab binding to ROS-DNA. The results demonstrate that modification of lysyl residues paralleled loss in Mab binding to ROS-DNA to the extent of 73%, suggesting the probable role of these positively charged amino acid residues in the complementarily determining regions of the Mab. The Mab was also used as an immunochemical probe to detect oxidative DNA damage in vivo in SLE. The Mab distinctly recognized five DNA samples out of eight from SLE patients and gave maximum inhibitions of 57, 58, 63, 64 and 70% in inhibition assay, while not reacting with DNA from normal, healthy population which served as negative control. High recognition of DNA isolates from SLE patients by the Mab having preferential binding to ROS-modified epitopes indicates increased oxidative stress in these patients leading to DNA damage which may contribute to the induction of antibodies cross-reacting with native DNA (nDNA).

Binding of circulating antibodies to reactive oxygen species modified-DNA and detecting DNA damage by a monoclonal antibody probe.

Circulating antibodies in the sera of normal, healthy humans of different age groups to native DNA (nDNA) and reactive oxygen species modified DNA (ROS-DNA) was studied by competition ELISA. Sera from the young population (< 50 years of age) showed negligible levels of anti-DNA antibodies. In contrast, anti-DNA antibodies were found in three sera from the moderately aged group (50-59 years) with a high binding to ROS-DNA (43-47%). In the aged group (> 60 years), four sera showed higher recognition of ROS-DNA (> 60% inhibition) over nDNA (55-60%). Oxidative lesions in human genomic DNA were immunochemically detected using the monoclonal anti-ROS-DNA antibody as a probe. The antibody has a high specificity for ROS-DNA and preferentially recognizes ROS-modified epitopes on nucleic acids. The study indicates low recognition of DNA isolated from the younger population, while in the age group 50-59 years, one DNA isolate showed a high inhibition (57%) in monoclonal antibody binding. Four DNA isolates from the aged group showed substantial inhibition in antibody activity to the extent of 49, 53, 64 and 69%. The results demonstrate an age-related increase in the levels of anti-DNA antibodies, with a higher recognition and binding to ROS-DNA. A high reactivity of DNA isolated from aged individuals by the monoclonal antibody indicates increased oxidative stress leading to DNA damage. It is suggested that free radical damage to DNA in vivo, particularly in the aged, alters its antigenicity and stimulates an immune response against modified DNA. These antibodies are cross-reactive to nDNA.

Immunochemical detection of oxidative DNA damage in cancer and aging using anti-reactive oxygen species modified DNA monoclonal antibody.

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions and ischemia. These species have been implicated in various disorders, mutagenesis, carcinogenesis and aging. Of many macromolecules, DNA is the most susceptible to hydroxyl radical, the most reactive of the ROS. The present study is designed to detect oxidative DNA damage in cancer patients and healthy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified calf thymus DNA fragments (approximate size 400 bp) were modified with OH, generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline sucrose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in Tm, modification of thymine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was characterized for antigen binding specificity by competition ELISA. Monoclonal antibody showed strong binding to ROS-modified DNA, its modified fragments, polynucleotides and bases. With the exception of native DNA, binding of unmodified polynucleotides and bases was much lower. The mAb distinctly recognized DNA samples from lymphocytes of healthy aged humans and gave maximum inhibitions of 49, 53, 64 and 70%, while not reacting with DNA from young population. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as negative control. The studies demonstrate that the mAb, although cross-reactive, preferentially binds ROS-modified epitopes on DNA. High reactivity of mAb to DNA samples from cancer patients and healthy aged humans indicates increased oxidative stress leading to DNA damage.

Reactive oxygen species modified thymine and poly(dT) present unique epitope for human anti-DNA autoantibodies.

Hydroxyl radical, one of the most potent of all reactive oxygen species has been implicated in many human degenerative diseases and is known to modify adenine and thymine in cellular DNA. In the present studies, adenine, thymine and their synthetic homopolymers poly(dA), poly(dT) were ROS-modified and subsequently used as inhibitors of native DNA binding to human anti-DNA autoantibodies. Besides nDNA, modified thymine and poly(dT) were effective inhibitors of DNA-anti-DNA antibody interaction. The relative affinity of ROS-modified poly(dT) was better than that of native DNA. Visual detection of modified thymine and poly(dT) binding to affinity purified anti-DNA IgG by an indirect band shift assay support competition inhibition data. The enhanced recognition of ROS-DNA by anti-DNA autoantibodies, as reported earlier, could be due to the ROS-induced modification of thymine.

Mutagenic and genotoxic activities of four pesticides: captan, foltaf, phosphamidon and furadan.

The mutagenic and genotoxic potential of four pesticides viz. captan, foltaf, phosphamidon and furadan was evaluated by the Ames mutagenicity assay and their DNA damaging ability on radiation repair defective E. coli K-12 strains respectively. The mutagenic spectrum revealed captan to be most mutagenic in the absence of metabolic activation, while the presence of S9 mix led to an attenuated mutagenic response. Foltaf, phosphamidon and furadan were detected as relatively weaker mutagens. A significant decrease in the survival of SOS defective mutants, recA, lexA and pol- of E. coli was observed as compared to their wild-type counterparts in the presence of the pesticides. The role of SOS repair genes gains further support from the Salmonella strains triggering the error-prone SOS response.

Anti-ROS-DNA monoclonal antibody as molecular probe for oxidative DNA damage.

Modification of 400 bp (approximate size) calf thymus DNA with OH radical resulted in lowered Tm, modification of thymine (58.3%), guanine (20%) and single strand breaks. Monoclonal antibodies (mAb) generated against ROS-DNA were of IgG1 subclass. The mAb showed strong binding to ROS-DNA and ROS-modified bases and polymers, in particular, of thymine. The mAb, therefore, preferentially recognizes ROS-modified epitopes on nucleic acids. Distinct binding to DNA isolated from aged, but not from normal humans by the monoclonal antibody was observed. The antibody effectively recognized oxidative lesions in DNA from cancer patients. These studies demonstrate the potential application of the mAb as an immunochemical probe to detect oxidative DNA lesions.

Isolation and characterization of four polycyclic aromatic hydrocarbon degrading bacteria from soil near an oil refinery.

Four bacterial strains (I-IV) capable of optimum growth on 0.1% naphthalene, anthracene or a mixture of naphthalene and phenanthrene were isolated from soil near an oil refinery. Two isolates (I and II) were identified as belonging to the genus Micrococcus, while strains III and IV were identified as Pseudomonas and Alcaligenes respectively. All the isolates were found to bear high molecular weight plasmid DNA (isolate I and IV 89%, II 67.5% and III 92.1% of lambda DNA), which is presumed to aid in the metabolism of polycyclic aromatic hydrocarbons. The strains also showed appreciable growth at high concentrations of NaCl (up to 7.5%).

Biodegradation of polycyclic aromatic hydrocarbons in soil around Mathura oil refinery, India.

Six polycyclic aromatic hydrocarbons [naphthalene, anthracene, phenanthrene, pyrene, chrysene and benzo(a)-pyrene] were detected in soil receiving effluents from an oil refinery. Biodegradation studies revealed a time-dependent disappearance of these polycyclic aromatic hydrocarbons when they were added to soil samples: naphthalene disappeared completely in 60 days, whereas phenanthrene, anthracene, pyrene, chrysene and benzo(a)pyrene decreased by 87%, 34%, 21%, 5% and 40%, respectively, in 120 days.