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M V Reddy

Publications and source records attributed to M V Reddy.

At least 73 records · Page 4Linked to original sources
PubMed1990

A comparison of DNA adduct formation in white blood cells and internal organs of mice exposed to benzo[a]pyrene, dibenzo[c,g]carbazole, safrole and cigarette smoke condensate.

Measurement of tissue/cell DNA adducts represents a suitable monitor of carcinogen exposure because the majority of chemical mutagens/carcinogens react with DNA, forming covalent adducts, a key event in the initiation of chemical carcinogenesis. Investigations of DNA-adduct formation in vivo in white blood cells (WBC) versus target tissues, i.e. internal organs for most carcinogens, is expected to yield useful information about the suitability of WBC for biomonitoring and risk assessment. For this purpose, female ICR mice were given 0.4 mmole/kg benzo[a]pyrene (BP), 0.045 mmole/kg dibenzo[c,g]carbazole (DBC) or 2.47 mmole/kg safrole by oral gavage or 4 daily doses (equivalent to 3 cigarettes) of cigarette-smoke condensate (CSC) by topical application. At 24 h after dosing, DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay [M.V. Reddy and K. Randerath, Carcinogenesis, 7 (1986) 1543] in WBC and internal tissues treated with individual carcinogens, while CSC treatment elicited aromatic adducts in most tissues but not in WBC. Adduct patterns of WBC DNA were qualitatively similar to those of internal organs, but adduct amounts varied. BP, a systemic carcinogen, bound nearly as much to WBC DNA as to target-tissue DNA samples; whereas the liver carcinogens, DBC and safrole, bound to WBC DNA considerably less (22- and 51-fold, respectively) compared with liver DNA. The number of adducts in 10(7) nucleotides of WBC, liver, lung, kidney and spleen DNA, respectively, were: 2, 5, 3, 2 and 3 with BP; 6, 131, 6, 14 and 4 with DBC; 5, 238, 3, 5 and 0.6 with safrole. For CSC, these values were 0, 1 and 0.02 in WBC, lung and spleen, respectively. Our results show that carcinogen binding to WBC DNA does not reflect binding to target-tissue DNA in a quantitative sense for the carcinogens studied except for BP, and that WBC are not suitable surrogates for monitoring CSC exposure by DNA-adduct measurement after topical application. The CSC data in mice was consistent with the previous findings in humans that smokers' tissues but not WBC show smoking-related bulky/aromatic DNA adducts, as measured by 32P-postlabeling.

Animals↗
PubMed1990

Diagnostic use of polyclonal antibodies raised in mouse ascitic fluid in bancroftian filariasis.

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA, 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) Sag antibody, 93% of microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

Animals↗
PubMed1990

32P-postlabeling assay for carcinogen-DNA adducts: description of beta shielding apparatus and semi-automatic spotting and washing devices that facilitate the handling of multiple samples.

The utilization of the 32P-postlabeling assay in combination with TLC for the sensitive detection and estimation of aromatic DNA adducts has been increasing in the past few years. The procedure consists of 32P-labeling of carcinogen-adducted 3'-nucleotides in the DNA digests using [gamma-32P]ATP and polynucleotide kinase, separation of 32P-labeled adducts by TLC, and their detection by autoradiography. During both 32P-labeling and initial phases of TLC, a relatively high amount of [gamma-32P]ATP (3.0-4.5 mCi) is handled when 30 samples are processed simultaneously. We describe the design of acrylic shielding apparatus, semi-automatic TLC spotting devices, and devices for development and washing of multiple TLC plates, which not only provide substantial protection from exposure to 32P beta radiation, but also allow quick and easy handling of a large number of samples, thus expediting the assay workup and making it less labor-intensive. Specifically, the equipment includes: (i) a multi-tube carousel rack (7.5 cm diameter and 7.7 cm height) having 15 wells to hold capless Eppendorf tubes (0.5 ml) and a rotatable lid with an aperture to access individual tubes; (ii) a pipet shielder; (iii) two semi-automatic spotting devices to apply radioactive solutions to TLC plates; (iv) a multi-plate holder for TLC plates; and (v) a mechanical device for washing multiple TLC plates. Item (i) is small enough to be held in one-hand, vortexed, and centrifuged to mix the solutions in each tube while beta radiation is shielded. Items (iii) to (iv) aid in the automation of the assay.

Carcinogenicity Tests↗
PubMed1990

DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates.

The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.

Acid Phosphatase↗
PubMed1990

Postlabeling and immunoassay analysis of polycyclic aromatic hydrocarbons--adducts of deoxyribonucleic acid in white blood cells of foundry workers.

Blood samples were obtained from volunteers who were occupationally exposed to polycyclic aromatic hydrocarbons in a Finnish iron foundry and from referents not known to be occupationally exposed to this class of chemical carcinogens. Aromatic adducts were determined in the deoxyribonucleic acid of white blood cells from the exposed workers with the 32P-postlabeling and immunologic techniques. There was a correlation between the estimated exposure in a particular job and the adduct levels. Jobs of men with high adduct levels (greater than 1 adduct/10(7) nucleotides in the postlabeling assay) included sand preparation, molding, shake-out, and transport. The adduct levels were low in men in pattern making, melting, and fettling. This study suggests that 32P-postlabeling and immunoassay may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

Benzo(a)pyrene↗
PubMed1990

Comparison of Dot-ELISA with Sandwich ELISA in detecting circulating antigen in patients with bancroftian filariasis.

Dot-ELISA assay was compared with standard Sandwich ELISA in detecting parasite antigen in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same serum samples were used in both assays. With Dot-ELISA, 67 of 70 serum samples from microfilaremic patients were positive at a dilution of 1:50. End titers ranged from 1:80 to 1:1 280. While with Sandwich ELISA, 64 of the 70 serum samples were positive at a dilution of 1:10. End titers ranged from 1:10 to 1:320. The specificity of both assays was over 91%, but their sensitivity was markedly different. Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human serum, whereas the lower limit of detection by Sandwich-ELISA was 10 ng/ml parasite antigen. An additional advantage of Dot-ELISA is that it does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.

Animals↗
PubMed1989

Wb E34 monoclonal antibody: further characterization and diagnostic use in bancroftian filariasis.

The Wb E34 monoclonal antibody raised against Wuchereria bancrofti microfilarial excretory secretory (mf ES) antigen was reported to be useful in detecting the filarial antigen in W. bancrofti and Brugia malayi infected sera. Further studies in this laboratory showed that this monoclonal antibody reacts with a stage-specific antigen of W. bancrofti filarial parasite. Wb E34 identified three antigenic components with molecular weights, 55, 57.5, and 63 kilodaltons (Kd) in western blot analysis. The target antigen of Wb E34 was found to be located in the cytoplasm of microfilariae by indirect immunofluorescence technique. The inhibitory antibodies to Wb E34 were detected in a higher percentage of microfilaraemic sera (81%) than in clinical filarial (33%) or tropical eosinophilia sera (35%). Thus the inhibition ELISA using W. bancrofti mf ES antigen and Wb E34-penicillinase may be useful in detecting the filarial antibody associated with the active stage of infection.

Animals↗
PubMed1989

A method for in vitro culture of rat Zymbal gland: use in mechanistic studies of benzene carcinogenesis in combination with 32P-postlabeling.

Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 micrograms/mL of benzene. Using a nuclease P1-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 10(9) nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 10(9) nucleotides), which decreased in the order benzoquinone greater than hydroquinone greater than phenol greater than benzenetriol greater than catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

2-Acetylaminofluorene↗
PubMed1989

32P analysis of DNA adducts in tissues of benzene-treated rats.

Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease P1-enhanced 32P-postlabeling assay, having a sensitivity limit of 1 adduct in 10(9-10) DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ), potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mg/kg, 5 days/week, in olive oil (3 mL/kg) for 1 day, 1 week, 5 weeks, and 10 weeks were analyzed by the 32P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per 10(9) DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to major adducts in vitro from the above specified compounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ, and BQ are either not formed in vivo with benzene or formed at levels below the detection limit of 1 adduct per 10(9-10) DNA nucleotides.

Animals↗
PubMed1988

Aromatic DNA adducts in white blood cells of foundry workers.

Blood samples were obtained from volunteers working in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons (PAH) and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene: high, greater than 0.2: medium, 0.05-0.2: low, less than 0.05 micrograms benzo[a]pyrene/m3 air). Aromatic adducts were found to be present in white blood cell DNA from most of the exposed workers using the enzyme-linked immunosorbent assay (ELISA) to detect aromatic DNA adducts and the 32P-postlabelling technique. There was a dose-response relationship between the estimated exposure and adduct levels by both methods, and a reasonable correlation between the results of the immunoassay and postlabelling carried out in two laboratories. The levels of adducts found in the samples from the high and medium exposure groups by ELISA ranged up to five adducts in 10(7) nucleotides: the aromatic adducts detected by the postlabeling assay were at a level of two adducts/10(8) nucleotides in the high and medium exposure categories. No effect due to age, sex or the smoking habits of the subjects was observed. The results indicate that DNA extracted from white blood cells of highly exposed workers is more likely to contain aromatic DNA adducts than that from workers without occupational exposure to PAH, but large interindividual variations were evident. This study suggests that the antibody and 32P-postlabelling assays may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C.

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.

Animals↗
PubMed1987

32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A 32P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10(7) to 10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are 32P-labeled at 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P1. The latter observation has been utilized to enhance the 32P-assay's sensitivity to 1 adduct in 10(10) nucleotides for a 10-micrograms DNA sample by postincubation of DNA digests with nuclease P1 before 32P-labeling. The enzyme dephosphorylates the normal nucleotides but not most aromatic and bulky nonaromatic adducts, so that only the latter serve as substrates for the kinase-catalyzed labeling reaction. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and in humans.

Animals↗
PubMed1987

Parasite antigens in sera and urine of patients with bancroftian and brugian filariasis detected by sandwich ELISA with monoclonal antibodies.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigens in human lymphatic filariasis. The assay utilizes a polyclonal rabbit antifilarial antiserum to capture, and a monoclonal antibody to identify, circulating parasite antigen. Using this assay, we found that greater than 95% of sera from microfilaremic donors with bancroftian or brugian filariasis, approximately 60% of sera from microfilaremic patients with hydroceles, chyluria, or elephantiasis, and 15%-20% of sera from asymptomatic residents of filariasis-endemic areas evidently contain filarial antigens. Antigen was also detected in the urine of some microfilaremic patients. Serum levels of antigen detected with one monoclonal antibody, ES34, correlated well with microfilarial density in night blood. In contrast, less than 5% of sera from residents of areas where lymphatic filariasis is not endemic reacted in the assay, even though approximately one-third of the donors whose sera were tested were known to be infected with intestinal nematodes. The assay was designed to be flexible enough to allow the parallel use of multiple monoclonal antibodies with different specificities and simple enough to be applicable in most areas where lymphatic filariasis is endemic.

Antibodies, Monoclonal↗
PubMed1986

Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts.

Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10(8)-10(10) DNA nucleotides). A 32P-postlabeling assay for detection and quantitation of carcinogen--DNA adducts with a sensitivity limit of 1 adduct in 10(7)-10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by t.l.c., detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in approximately 10(10) nucleotides for a 10-micrograms DNA sample. Nuclease P1 cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonucleosides which do not serve as substrates for polynucleotide kinase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease P1. The additional enzymatic step enabled specific labeling of adducts in up to 20 micrograms of DNA with excess carrier-free [gamma-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7,12-dimethyl-benz[a]anthracene, dibenzo[c,g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure.

9,10-Dimethyl-1,2-benzanthracene↗