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[An experimental study on uptake of Cr(51) tagged red blood cells to Paragonimus westermani in definitive host]

Authors carried out the experimental study to investigate whether the Paragonimus westermani would uptake the blood in the parasitic definitive host, and also tried for the amount and distribution of the blood which would be harbored in a worm body, and so on. Experimental animals used in this study were dogs and cats, and they were infected with metacercariae of P. westermani. Several months later of the infection, Cr(51) tagged red blood cells were administered to each animal, and the animals were sacrificed and autopsy was done after 24 or 48 hrs of the administration. One part of the detected P. westermani adult worms of each animal were bred in Tyrode's solution and the others were killed by fixing in formalin solution. By Well-type scintillation counter, the radioactivities were measured for the alive and killed worms and of the blood of each experimental animal which was collected at autopsy. In conclusion, it was found that P. westermani worms uptake blood in parasitic definitive host, and amount of the blood harboring in a worm of 56 infection-days(size of worm; 4 x 3 x 1 mm) was 0.002 ml, 0.004 or 0.005 ml in 103 infection-days(size of worm: 8 x 5 x 3 mm) and 0.011 ml in 217 infection-days(size of worm: 9 x 7 x 4 mm). Almost of the whole blood uptaked in the worms was distributed in intestine. It was observed that the blood uptaked in the worm body was excreted continuously through the excretory pore, so that the radioactivity could not measured almost completely in cases of 24 hrs-breeding worms.

Journal Article↗

[Cytotoxicity of resident and lymphokine-activated mouse peritoneal macrophage against Trichomonas vaginalis].

This study was aimed to observe the direct and lymphokine-activated cell mediated cytotoxic effects against Trichomonas vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured as release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. A 0.1 ml suspension of labeled protozoa (2 x 10(5)/ml) was placed in each well, followed by 0.1 ml of a suspension containing increasing numbers of peritoneal cells. After a 24 hr incubation at 37 degrees C, 0.1 ml of the supernatant was collected and counted in liquid scintillation counter. Mouse peritoneal macrophages had appreciable level of spontaneous cytotoxicity against T. vaginalis at the effector to target cell ratios from 5:1 to 50:1. Treatment of macrophages with lymphokine, produced by PHA-stimulated spleen cells, increased the cytotoxicity in comparison with resident macrophages against T. vaginalis. The degree of macrophage activation for the killing was not dependent upon the lymphokine concentration. Peritoneal cells adherent to plastic displayed significant levels of cytotoxicity against T. vaginalis. This study indicates that mouse peritoneal macrophages are spontaneously cytotoxic for T. vaginalis and lymphokine increases the cytotoxicity by activating macrophages to kill T. vaginalis.

Animals↗

Reversal of multidrug resistance in drug-resistant human gastric cancer cell line SGC7901/VCR by antiprogestin drug mifepristone.

AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. METHODS: Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcription-polymerase chain reaction(RT-PCR). Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2, Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells. Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis. The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular [(3) H]VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively. RESULTS: Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively. After treatment with 1, 5, 10, and 20 micromol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively. MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells. The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose. After 96 h of incubation with mifepristone 10 micromol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+/-0.65)% from (25.32+/-1.44)%, whereas the expression of Bax protein was increased to (19.69+/-1.13)% from (1.24+/-0.78)% (P<0.01). Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis. CONCLUSION: Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Study on immune function of dendritic cells in patients with esophageal carcinoma.

AIM: To investigate the immune function of dendritic cells from both peripheral blood and operated tissues of esophageal carcinoma patients in order to find the relationship between the immune function of dendritic cells and the pathogenesis of esophageal carcinoma. METHODS: The expression of CD83, CD80, and CD86 on the surface of dendritic cells cultured from the peripheral blood of patients was detected compared with that from health donors using flow cytometry. The ability of dendritic cells to induce T lymphocyte proliferation was evaluated by a liquid scintillation counter. The expression of CD80, CD86, CD83, and S-100 proteins was assessed in esophageal carcinoma tissues using immunohistochemical method. RESULTS: Compared with those from healthy donors, dendritic cells cultured from the peripheral blood of patients expressed lower CD80 and CD86. Furthermore, the ability of dendritic cells in patients to induce T lymphocyte proliferation was significantly lower than that of the control group. Compared with the control group, the positive expression ratio and frequencies of CD80, CD86, and S-100 in esophageal carcinoma tissues were significantly down regulated. The expression of CD83 was up-regulated in the pericancerous tissues, but no expression was found in the cancerous nodules. CONCLUSION: The impaired immune function and the decreased number of dendritic cells cause pathogenesis and progression of esophageal carcinoma.

Aged↗

Macrophage migration inhibitory factor stimulated by Helicobacter pylori increases proliferation of gastric epithelial cells.

AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2), its three isogenic mutants (TN2Deltacag, TN2DeltacagA and TN2DeltacagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of 3H-thymidine, and the levels of incorporation of 3H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2DeltacagA and TN2 DeltacagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2delta cag did not increase the release of MIF at any of the four ratios. 3H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2DeltacagA and TN2DeltacagE, but not from the mutant TN2Deltacag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H pylori-induced gastric epithelial cell proliferation.

Antibodies, Monoclonal↗

Influence of methionine/valine-depleted enteral nutrition on nucleic acid and protein metabolism in tumor-bearing rats.

AIM: To investigate the effects of methionine/valine-depleted enteral nutrition (EN) on RNA, DNA and protein metabolism in tumor-bearing (TB) rats. METHODS: Sprague-Dawlley (SD) rats underwent jejunostomy for nutritional support. A suspension of Walker-256 carcinosarcoma cells was subcutaneously inoculated. 48 TB rats were randomly divided in 4 groups: A, B, C and D. The TB rats had respectively received jejunal feedings supplemented with balanced amino acids, methionine-depleted, balanced amino acids and valine-depleted for 6 days before injection of 740 KBq (3)H- methionine/valine via jejunum. The (3)H incorporation rate of the radioactivity into RNA, DNA and proteins in tumor tissues at 0.5, 1, 2, 4 h postinjection of tracers was assessed with liquid scintillation counter. RESULTS: Incorporation of (3)H into proteins in groups B and D was (0.500+/-0.020) % to (3.670+/-0.110) % and (0.708+/-0.019) % to (3.813+/-0.076) % respectively, lower than in groups A ((0.659+/-0.055) % to (4.492+/-0.108) %) and C ((0.805+/-0.098) % to (4.180+/-0.018) %). Incorporation of (3)H into RNA, DNA in group B was (0.237+/-0.075) % and (0.231+/-0.052) % respectively, lower than in group A (P<0.01). There was no significant difference in uptake of (3)H by RNA and DNA between group C and D (P>0.05). CONCLUSION: Protein synthesis was inhibited by methionine/valine starvation in TB rats and nucleic acid synthesis was reduced after methionine depletion, thus resulting in suppression of tumor growth.

Amino Acids↗

[Affinity of the elements in group VI of the periodic table to tumors and organs].

In order to investigate the tumor affinity radioisotopes, chromium (51Cr), molybdenum (99Mo), tungsten (181W), selenium (75Se) and tellurium (127mTe)--the elements of group VI in the periodic table--were examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. Seven preprarations, sodium chromate (Na251CrO4), chromium chloride (51CrCl3), normal ammonium molybdate ((NH4)299MoO7), sodium tungstate (Na2181WO4), sodium selenate (Na275SeO4), sodium selenite (Na275SeO3) and tellurous acid (H2127mTeO3) were injected intravenously to each group of tumor bearing rats. These rats were sacrificed at various periods after injection of each preparation: 3 hours, 24 hours and 48 hours in all preparations. The radioactivities of the tumor, blood, muscle, liver, kidney and spleen were measured by a well-type scintillation counter, and retention values (in every tissue including the tumor) were calculated in percent of administered dose per g-tissue weight. All of seven preparations did not have any affinity for malignant tumor. Na251CrO4 and H2127mTeO3 had some affinity for the kidneys, and Na275SeO3 had some affinity for the liver. Na2181WO4 and (NH4)299MoO4 disappeared very rapidly from the blood and soft tissue, and about seventy-five percent of radioactivity was excreted in urine within first 3 hours.

Animals↗

[Study of 67Ga distribution in neoplasms and in liver by a cell fractionation method].

Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of lysosome in accumulation of 67Ga in malignant tumor tissue and liver. The following animals and transplanted tumors were used: rats implanted with Yoshida sarcoma and hepatoma AH109A; mice implanted with Ehrlich tumor. 67Ga-citrate were injected to the rats intravenously and to the mice intraperitoneally. Ten minutes to 48 hours after the administration of 67Ga-citrate, the animal were sacrificed, and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and livers were carried out according to the method of Hogeboom and Schneider. Radioactivity of each fraction was counted by a well type scintillation counter, and protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and small amount of radioactivity was localized in the mitochodrial fraction (lysosome contains in this fraction). But in the liver, most of the radioactivity was concentrated in the mitochondrial fraction and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of mitochondrial fraction was increased with the passage of time after administration, and about 30% of total activity was concentrated in this fraction at 24 hours after administration. From these results it is concluded that lysosome doses not play an important role in the tumor concentration of 67Ga and lysosome plays an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A it is presumed that lysosome plays considerably important role in the tumor concentration of 67Ga, hepatoma AH109A having some nature of liver.

Animals↗

[Experimental study on attachment of bacillus Calmette-Guerin to bladder wall with different types of injury].

OBJECTIVE: To investigate whether different types of injury on bladder wall can influence bacillus Calmette-Guerin (BCG) attachment. METHODS: The bladder mucosa of 24 rabbits were treated by electrocautery,cryocautery and incision on left lateral wall, right lateral wall and posterior wall, respectively. Then radiolabeled BCG ((3)H-BCG) was instilled into bladder. Two hours latter, the injured bladder wall with different methods and non-injured wall (anterior wall of bladder) were surgically removed and digested. The quantity of BCG of each specimen was determined by liquid scintillation counter. RESULT: The quantity of BCG attachment to bladder wall with different injuries was significantly higher than that of non-injured wall (P<0.001), meanwhile there was no statistically difference among the BCG levels of different injury types (P>0.05). CONCLUSION: BCG attachment is not influenced by different types of injury on the bladder wall.

Animals↗

[Identification of sentinel lymph node in neck-node-negative oral and pharyngeal carcinoma study of patients, it's feasibility, and problems].

We assessd the feasibility and problems associated with sentinel lymph node (SLN) study in 13 cases of oral and pharyngeal squamous cell carcinoma (SCC) that were neck-node-negative clinically. The primary sites were the tongue (n = 10), other sites in the oral cavity (n = 2), and the mesopharynx (n = 1). The day before surgery, tracer was injected into the submucosa around the tumor, and scintigraphic images were acquired 2 hours later. The SLN was identified intraoperatively with a handheld gamma probe, and neck dissection, including the SLNs, was performed. Radioactivity within the nodes was confirmed with a well type scintillation counter, and all resected lymph nodes were histologically examined for metastasis. The SLN was identified in every case. There were regional lymphnode metastases in 4 cases, and metastasis to the SLNs was present in all of 4 cases. Thus, the SLN concept was valid for head and neck SCC, sentinel node navigation surgery (SNNS) was thought to be applied in stage NO SCC of the head and neck. If SNNS is performed, about 70% of patients do not require neck dissection. SNNS is feasible and cost-effective in these cases. We used two different tracers: phytate and tin colloid, and found that phytate was more useful. To avoid the effects of the shine-through phenomenon, it was thought that some directions of lymphoscintigram should be taken. For intraoperative identification of the SLNs, care should be taken to the angle of gamma probe. SLN study leads to clarify each patient's lymphoid flow mapping, and it is also useful to determine the dissection area of selective neck dissection.

Adult↗

[The analysis of the difference in anti-cancer drug sensitivity of 3 clones separated from bladder cancer cell line].

One of the major problems with cancer chemotherapy is the development of drug resistance during treatment. Two mechanisms are considered as the cause of drug resistance, natural and acquired. It is now considered that cancers can be composed of multiple clonal subpopulations of cancer cells. In this study, we separated three clones (C1, C3 and C8) from NBT-2 (human bladder cancer cell line) by limiting dilution. We examined the growth rate and the transplantability to nude mice and performed chromosomal analysis of three clones. The doubling time of C1 is 22 hours, and those of C3 and C8 were 25 and 36 hours, respectively. Each clone was transplantable to nude mice, but we could not find out any histological difference among them. The chromosome numbers of C1 was 66, and those of C3 and C8 were 68 and 63, respectively. We could also find out karyotypic difference among them. We could therefore consider that these three clones had different biological features and studied the difference in drug sensitivity among these three clones and the parent cell line. Cells (1 x 10(4)/well) were incubated in microplates with ten different chemotherapeutic agents for 72 hours. Then 3H-thymidine (1 microCi/ml) was added to each. After 24 hours, cells were harvested and the uptake of 3H thymidine was counted with a liquid scintillation counter. According to the reaction pattern, these chemotherapeutic agents were divided into three groups. 1. The radio isotope uptake of three clones and parent cell line was proportionally inhibited by increasing the drug concentration (carboplatin, (glycolato-o, o-) diammine platinum (II), ifosfamide).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Duchenne muscular dystrophy. Effects of vitamin E administration on urinary luminescence].

BACKGROUND: Urinary luminescence is increased in patients with Duchenne muscular dystrophy, probably due to the higher oxidative stress present in this disease. AIM: To assess the effects of vitamin E supplementation on urinary luminescence in children with Duchenne muscular dystrophy. PATIENTS AND METHODS: Eighteen children with muscular dystrophy aged 12.2 years old and nine control children aged 10 years old, received 400 IU/day of vitamin E during one month. Prior to supplementation and twice a week thereafter, spot urine samples were obtained to measure urinary luminescence in a scintillation counter. RESULTS: There was a wide variability in urinary luminescence within and between children. Mean values decreased after vitamin E supplementation in six of nine controls and in 12 of 18 children with muscular dystrophy. CONCLUSIONS: Vitamin E supplementation significantly decreases urinary luminescence in healthy children and in patients with Duchenne muscular dystrophy. Therefore, it could be useful for the treatment of this disease.

Adolescent↗

Inhibitory effects of Woodward's reagent K on carrier-mediated anion transport in rabbit intestinal brush border membrane vesicles.

BACKGROUND: Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate) is a negatively charged reagent that covalently and irreversibly modifies carboxyl-containing amino acid residues. Under conditions of an outwardly directed hydroxide (OH.) gradient (pHin > pHout), folate uptake by jejunal brush border membrane (BBM) vesicles is mediated by a folate:OH. exchanger or a phenomenologically indistinguishable proton (H+):folate cotransporter. Under these pH conditions, Woodward's reagent K rapidly inhibits folate uptake in a pH-, temperature- and concentration-dependent manner. METHODS: Rabbit BBM vesicles were prepared using the divalent cation precipitation method. Vesicle uptake of 3H-folate was measured by a rapid filtration technique. Radioactivity was gauged using a liquid scintillation counter. RESULTS: The initial rate of OH. gradient-stimulated folate uptake remained linear in the presence or absence of Woodward's reagent K, suggesting that Woodward's reagent K inhibition is not secondary to more rapid dissipation of the transmembrane pH gradient. Woodward's reagent K also inhibited other modes of transport mediated by the folate carrier, 3H-folate:folate exchanger, ileal sulfate (SO)4(2):OH. exchanger, but neither sodium (Na+):glucose, Na+:SO4(2.) nor Na(+):alanine cotransporters, were inhibited by 3 mM Woodward's reagent K. CONCLUSIONS: The inhibition of intestinal anion exchangers by Woodward's reagent K suggests a critical role for carboxyl group--containing amino acids in these transport processes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Isolation and partial characterization of rat liver porphyrin binding proteins.

14C-coproporphyrin III was added to hemoglobin-free cytoplasmic rat liver extract. Molecular sieving was performed on Sephadex G-75 column. The protein content of each fraction was measured at 280 nm in double beam spectrophotometer and the radioactivity by means of liquid scintillation counter. In several runs on Sephadex we found four main radioactive peaks which correlate to four porphyrin binding proteins in rat liver cytoplasm. One fraction, which probably is identical with Y protein or ligandin, was further purified and characterized by means of different chromatographic techniques.

Animals↗

The role of alpha1-adrenoceptors in the clonidine-induced contraction and relaxation of rat aorta.

Clonidine causes dilatation of the aorta in the presence of endothelium, while it causes contraction of the aorta in the absence of endothelium. The present study was carried out to clarify the role of alpha-1-adrenoceptors in the vascular action of clonidine. The aortic rings were suspended in Krebs-Henseleit (K-H) medium, and the effects of alpha-1- and alpha-2-adrenoceptor antagonists on the clonidine-induced contractions were measured. Moreover, the role of the phosphatidylinositol (PI) response was examined. The aortic slices were incubated in K-H medium containing, [3H]myo-inositol and clonidine. The formation of [3H]inositol monophosphate (IP1) was measured with a liquid scintillation counter. Clonidine caused contraction of the aorta in the absence of endothelium, in a dose-dependent manner. This contraction was inhibited by antagonists in the following order of the potency: prazosin > phentolamine > spiperone > urapidil = yohimbine > L-659066 > atipamezole. On the other hand, clonidine inhibited norepinephrine (NE)-induced contraction in the aorta in the absence and in the presence of endothelium. Clonidine enhanced IP1 accumulation in the aorta in the absence of endothelium, whereas it inhibited NE-induced IP1 accumulation in the aorta. The present results show that alpha-1-adrenoceptors are probably involved in the clonidine-induced contraction and relaxation of the rat aorta.

Adrenergic alpha-1 Receptor Agonists↗

[The 131I-fibrinogen turnover during activation of blood fibrinolysis].

The influence of stanozolol Stromba and phenformin Dibotin (Winthrop) and complamin (Wulfing) on transformation of 131J-fibrinogen administered intravenously was examined in 39 patients with arteriosclerosis obliterans. The patients were divided into 3 groups; one of the groups included untreated patients. Radioactivity of the plasma and of the urine was determined by means of a scintillation counter. The volume of plasma, plasma fibrinogen (g%, g, g/kg), half time of persistance (T 1/2) and degradation of fibrinogen were determined. The patients who received Dibotin and Stromba simultaneously showed statistically significant decrease of the plasma fibrinogen degradation im comparison to the control group. But in patients who received complamin in spite of significant decrease of the plasma fibrinogen (g%), the fibrinogen degradation was not different from the value obtained in the group.

Adult↗

Effect of catecholamic acid on detoxication and distribution of NiCl2 in mice and rats.

AIM: To study the effect of catecholamic acid (CBMIDA) on detoxication of NiCl2. METHODS: Mice and rats were injected s.c. or i.m. CBMIDA immediately after i.p. NiCl2. Each mouse was injected i.p. CBMIDA after i.v. 63NiCl2 185 kBq, and radioactivities of various tissues were measured with liquid scintillation counter at 24 h. The localization of 63Ni was shown by the whole-body autoradiography. RESULTS: CBMIDA s.c. 0.5-1.5 g.kg-1 markedly reduced the mortality from acute poisoning of i.p. NiCl2 500 mg.kg-1. After i.p. NiCl2 in mice, the LD50 was 82.7 mg.kg-1. Mice were injected s.c. CBMIDA 1.5 or 2.5 g.kg-1 after Ni poisoning, the LD50 of NiCl2 were raised to 789 or 820 mg.kg-1, respectively. The LD50 of NiCl2 was 39 mg.kg-1 in rat. If CBMIDA was injected i.m. 0.5 g.kg-1 after i.p. NiCl2, the LD50 was 332 mg.kg-1. CBMIDA 1.5 g.kg-1 i.m. after i.v. 63NiCl2, decreased the contents of 63Ni in blood and lung of mice vs control mice at 24 h. The contents of 63Ni in brain, heart, spleen, and kidney were similar to those of the control mice. The content of 63Ni in bone was more than the control. The excretions of 63Ni through urine and feces were not increased by CBMIDA at 24 h. The whole-body autoradiography showed that the radioactivity was highly localized in the kidney, lung, and Harder's gland. There was a moderate level of 63Ni in the liver, bone, skin, and blood. A pronounced accumulation occurred in the bone. There was a marked reduction of 63Ni in the lung, skin, liver, and blood after i.p. CBMIDA. CONCLUSION: The CBMIDA markedly raised the survival rate of nickel-poisoned mice and rats, and decreased 63Ni levels in lung and blood.

Animals↗

Chemiluminescence enzyme immunoassay of 8-oxoguanine in DNA.

A test system has been developed to determine 8-oxoguanine in DNA, the most important biomarker of damage to DNA bases by reactive oxygen species. The system is based on a chemiluminescence enzyme immunoassay with the use of monoclonal antibodies (mcAB) against 8-oxoguanine. The test involves several stages: 1) immobilization of DNA on nitrocellulose membrane filters using an efficient technique with preliminary formation of a complex with protamine sulfate; 2) formation of antigen--antibody complexes (mcAB with 8-oxoguanine in DNA) with secondary antibodies and with a peroxidase--antiperoxidase complex (PAP method); 3) detection of increased chemiluminescence in a solution of hydrogen peroxide, luminol, and p-iodophenol. The increased chemiluminescence is determined with a conventional liquid scintillation counter for measuring beta-radioactivity. The system was tested by determining 8-oxoguanine formation in DNA upon gamma-irradiation and upon photosensitized oxidation of guanine under visible light in the presence of methylene blue. A linear dose dependence of 8-oxoguanine formation in DNA was shown for gamma-irradiation. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecule per 100 eV) is convenient to use for calibration of the amount of 8-oxoguanine formed under other conditions. The sensitivity of the method permits the detection of several femtomoles of 8-oxoguanine in a 40 microg sample of DNA.

Animals↗