N-acetyl derivative as the major active metabolite of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole in rat bile

2-Amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) is a mutagen and carcinogen isolated from a glutamic acid pyrolysate. When this 14C-labeled compound was administered to male F344 rats at a dose of 0.3 mCi (20.8 mg)/kg b.w., 70% of the radioactivity was excreted into the bile in 24 h. On HPLC analysis of this bile, several metabolites of Glu-P-1 were found with unmetabolized Glu-P-1. One of the mutagenic metabolites was identified as N-acetyl-Glu-P-1. This metabolite had a specific mutagenic activity of about one quarter of that of Glu-P-1 and its amount in the bile corresponded to a few percent of the dose of Glu-P-1 administered.     

Changes of erythrocytes due to hyperoxygenation

After exposure of animals to pure oxygen for an 1--3 hrs daily for 2--8 weeks the reticulocyte concentration increased 1.5--5 times in cats and decreased 2--4 times in rabbits of different age. The hemoglobin affinity to oxygen increased and then returned to normal level in adult rabbits, but only decreased in cats. Blood parameters in newborn rabbits whose mothers were breathing pure oxygen within 4 weeks of gestation up to the parturition were closer to those of adult animals as compared to control newborn rabbits. The hemoglobin affinity to oxygen in hyperoxic animals to normal levels after dialysis. This points to the importance of the salt environment within the erythrocytes for changes of the hemoglobin affinity to oxygen in hyperoxic conditions.     

Effect of dipyridoimidazole pretreatment on mutagen activation in rats

Rats were pretreated with a single oral dose of different mutagenic fractions obtained from glutamic acid pyrolysate: Glu-P-2 (2-amino-dipyrido[1,2-a:3',2'-d]imidazole), Glu-P-3 (3-amino-4,6-dimethyldipyrido[1,2-a:3',2'-d]imidazole), the tar residue and a basic extract (B2). The liver S9 fractions of these animals were used to investigate the mutagenic activation of 3 promutagens (2-aminoanthracene, Glu-P-2 and Glu-P-3) in Salmonella typhimurium strain TA1538. Different factors were analyzed; influence of the structure of the compounds administered, doses, time interval between pretreatment and sacrifice and sex of the rats. Interpretation of the hepatic induction effects was complicated, however, by the fact that simple oral pretreatment with the solvents (DMSO or ethanol) enhances the activation of the substrates tested for mutagenicity. A dose-effect relationship was found between 2-AA mutagenic activation and Glu-P-2 pretreatment. Glu-P-3 induced the activation of 2-AA more than did Glu-P-2, in the male as in the female. The mutagenicity of 2-AA activated with S9 from male rats was found to be optimal after 24 h pretreatment with 20 mg Glu-P-2/kg b.w. The mutagenicity of Glu-P-2 was poorly influenced by the different pretreatments applied to either the males or the females, whereas some dose effect was found in the autoinduction of Glu-P-2 mutagenicity. Compared to Glu-P-2, the mutagenicity of Glu-P-3 was increased at higher levels when tested with S9 from males pretreated with the same compound, but no differences were observed between males and females.     

Red blood cell phagocytosis and lysis following oxidative damage by phenylhydrazine

Red blood cells exposed in vitro to phenylhydrazine acquired Heinz bodies, bound autologous IgG and were then phagocytized when incubated with autologus mononuclear phagocytes. In vivo, phenylhdyrazine administered to rabbits, caused the appearance of high plasma hemoglobin levels and hemoglobinuria as well as Heinz body formations and IgG binding to erythrocytes. This suggests that while in vitro the main mechanism of red cell removal seems to be phagocytoses, in vivo both intravascular hemolysis and phagocytosis are active processes. Preliminary biochemical studies on phenylhydrazine-exposed erythrocytes showed that together with the well-known appearance of Heinz bodies, methemoglobin and a drop in reduced glutathione, this drug also causes ATP depletion. This is initially concomitant with the appearance of ADP and AMP and subsequently hypoxantine. Thus, irreversible ATP depletion may contribute to the genesis of the hemolytic process observed in vivo.     

The formation of heart DNA adducts in F344 rat following dietary administration of heterocyclic amines

Most heterocyclic amines formed during the cooking of meat and fish have been shown to form adducts in the livers of rats. Recently, however, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), administered in the diet to Fischer 344 (F344) rats for 4 weeks, was shown to produce the highest levels of adducts in the heart. In the present study 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-6-methyldipyrido[1,2-a:1',2'-d]imidazole (Glu-P-1) were given to F344 rats at carcinogenic dose levels (IQ 0.03%, MeIQx 0.04%, Trp-P-1 0.015%, Glu-P-1 0.05%) in the diet for 4 weeks. DNA adducts in the liver and heart were analyzed by 32P-postlabeling. DNA adducts were demonstrated to appear in the hearts of all animals exposed to heterocyclic amines at the following levels: IQ, 1.8 adducts/10(7) nucleotides, MeIQx, 3.8/10(7) ntd, Trp-P-1, 20/10(7) ntd and Glu-P-1, 7.2/10(7) ntd. Values for the heart were 10-20% of the respective liver adduct levels. Heart adducts increased linearly throughout the observed period when MeIQx was administered for up to 40 weeks. When MeIQx feeding was discontinued after 20 weeks and the animals subsequently given the basal diet, the adduct level at 20 weeks did not change during the following 20 weeks. A possible role for heart DNA alterations caused by food-borne heterocyclic amines in the development of age-related myopathies and cardiovascular disease is not inconceivable.     

Beneficial effect of succinic acid monoethyl ester on erythrocyte membrane bound enzymes and antioxidant status in streptozotocin-nicotinamide induced type 2 diabetes

Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment of non-insulin dependent diabetes mellitus. In the present study the effect of EMS and metformin on erythrocyte membrane bound enzymes and antioxidants activity in plasma and erythrocytes of streptozotocin-nicotinamide induced type 2 diabeteic model was investigated. Succinic acid monoethyl ester was administered intraperitonially for 30 days to control and diabetic rats. The effect of EMS on glucose, insulin, hemoglobin, glycosylated hemoglobin, TBARS, hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (Gpx), glutathione-S-transferase (GST), vitamins C and E, reduced glutathione (GSH) and membrane bound enzymes were studied. The effect of EMS was compared with metformin, a reference drug. The levels of glucose, glycosylated hemoglobin, TBARS, hyderoperoxide, and vitamin E were increased significantly whereas the level of insulin and hemoglobin, as well as antioxidants (SOD, CAT, Gpx, GST, vitamin C and GSH) membrane bound total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase were decreased significantly in streptozotocin-nicotinamide diabetic rats. Administration of EMS to diabetic rats showed a decrease in the levels of glucose, glycosylated hemoglobin, lipid peroxidation markers and vitamin E. In addition the levels of insulin, hemoglobin, enzymic antioxidants, vitamin C, and GSH and the activities of membrane bound enzymes also were increased in EMS and metformin treated diabetic rats. The present study indicates that the EMS possesses a significant beneficial effect on erythrocyte membrane bound enzymes and antioxidants defense system in addition to its antidiabetic effect.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

Quantifying adductive modification of hemoglobin from mice exposed to benzo[a]pyrene

The present work describes a method for the detection of minute amounts of benzo[a]pyrene, as the diolepoxide metabolite, bound covalently to the hemoglobin of erythrocytes isolated from mice previously exposed to the carcinogen. The technique consists of the acid-induced removal of the pyrenyl moiety from the hemoglobin as the strongly fluorescent free tetrols and their isolation by bonded-phase extraction methods and subsequent quantitation by fluorescence/HPLC. With this procedure as little as 5 pg of tetrol can be detected. The assay was used to determine the amount of benzo[a]pyrene-hemoglobin adduct formation in mice bearing a carcinogen-induced fibrosarcoma.     

The research background for risk assessment of ethylene oxide: aspects of dose

Data for relationships between in vivo doses inferred from levels of hemoglobin (Hb) or DNA adducts and administered (by inhalation or injection) doses of ethylene oxide (EO) in mice, rats and humans are reviewed. At low absorbed doses or dose rates these relationships appear to be linear, whereas at higher dose rates deviations from linearity due to saturation kinetics of detoxification and of DNA repair as well as certain toxic effects have to be allowed for. If these factors are taken into consideration, a rather consistent picture is obtained for animal studies, with a variation by less than a factor 2 between estimates of adduct level increments or in vivo dose increments per unit of administered dose. Although the value for in vivo dose per unit of exposure dose (ppm-hour) in humans is uncertain because of unreliable data for the time-weighted average exposure level, the most likely value for this relationship, supported by data for ethene, agrees with data for the rodents. In the animal species testis doses are approximately one-half of the blood doses inferred from Hb adducts.     

The interaction of phenyldichloroarsine with erythrocytes

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 mumol PDA/10(9) cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 mumol/10(9) cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd approximately 5 microM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.     

聚合牛血红蛋白抗原性的初步研究

对乙醇醛聚合的牛血红蛋白的抗原性进行了研究。将聚合的牛血红蛋白桉兔血量的10％和20％分别输入兔耳缘静脉,间隔7天后重复输液,共输注3次。ELISA检测未显示抗体产生。将聚合的牛血红蛋白、人血红蛋白和兔血红蛋白分别与免疫佐剂混合常规免疫家兔3次,并用上述抗原包被聚乙烯板,用ELISA方法检测抗体滴度,结果显示聚合牛血红蛋白和人血红蛋白加佐剂免疫家兔后均产生抗体,且有交叉反应,表明这两种血红蛋白有同源性,存在相似的抗厚决定簇。兔血红蛋白免疫家兔后无抗体产生,且无交叉反应,表明机体对自体蛋白有天然的耐受。     

Detection of N-acetyl derivative of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) in Glu-P-1-injected rats

In order to confirm in vivo N-acetylation of 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) in rats, we developed a new high-performance liquid chromatography (HPLC) method for detecting the N-acetyl derivative of Glu-P-1 in animal organs. Using this method, N-acetyl-Glu-P-1 was detected in rat liver, kidney and intestinal contents 6 h after intraperitoneal injection of Glu-P-1. This fact provides evidence to support in vivo N-acetylation of Glu-P-1 in rats.     

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.     

Distribution along DNA of the bound carcinogen N-acetoxy-N-2-acetylaminofluorene in chromatin modified in vitro.

Chromatin from duck erythrocytes was modified in vitro by the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Ac-O-AAF). The distribution of the carcinogen along the DNA molecule was studied using staphylococcal nuclease which allows the fractionation of chromatin DNA into two zones. It was shown that the carcinogen binds preferentially to the regions of chromatin sensitive to the enzyme; however, the regions of DNA tightly bound to histones and resistant to the enzyme react comparatively well. The single-strand specific nuclease S1 which digests DNA modified by the carcinogen in vitro did not digest chromatin under the conditions used. Some possible mechanisms for the interaction of the carcinogen with chromatin are discussed.     

Effect of 5-Campestenone (24-methylcholest-5-en-3-one) on Zucker diabetic fatty rats as a type 2 diabetes mellitus model.

We examined the therapeutic effects of dietary exposure to 5-campestenone (24-methylcholest-5-en-3-one), an enone derivative of campesterol, in Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes mellitus. Dietary 0.6 % exposure to 5-campestenone caused marked reduction in hemoglobin A1c (HbA1c), plasma total cholesterol, triglycerides and non esterified fatty acid (NEFA). In particular, plasma triglyceride levels were reduced in the 0.6 % 5-campestenone-fed group to about 25 % of that in the control group. In the oral glucose tolerance test (OGTT) at three and seven weeks after the beginning of treatment, 5-campestenone limited the rise of blood glucose levels by oral administration of glucose dose-dependently. Amounts of adipose tissue in the retroperitoneum and periepididymal area as well as abdominal subcutaneous fat were significantly decreased in animals fed 0.6 % 5-campestenone. The blood leptin concentration on the final day of feeding was significantly in animals administered 5-campestenone. No obvious anomaly due to consumption of 5-campestenone was detected in necropsy or clinical observations.     

Metabolic activation of a mutagen, 2-amino-6-methyldipyrido-[1,2-a:3′,2′-d]imidazole. Identification of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole and its reaction with DNA

A potent mutagen, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(Glu-P-1), isolated from pyrolysates of L-glutamic acid and casein, was metabolically activated and bound to DNA. An activated form was identified as 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(N-OH-Glu-P-1). Synthetic N-OH-Glu-P-1 reacted with DNA only after O-acetylation to give a modified DNA, which on hydrolysis gave 2-(C⁸-guanyl)amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole(gua-Glu-P-1). The same adduct was isolated from DNA modified with Glu-P-1 by microsomes in vitro, as reported earlier.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Effects of pyridoxine supplementation on blood glucocorticoids level, aspartate aminotransferase activity and glucose tolerance pattern under acute hypoxic stress

Studies were conducted on male adult rabbits to find out the changes in blood glucocorticoid levels along with the changes in aspartate aminotransferase activity in blood and the role of pyridoxine on the glucose tolerance pattern under hypoxic stress. Hypoxic stress was produced by exposing the animals to a simulated altitude of 7,000 m for 6 h. In the first set of experiments 10 rabbits were used. Blood haemoglobin level, plasma and erythrocyte glucocorticoid levels and erythrocyte GOT activity were measured just before and after the exposure to hypoxia. Erythrocyte GOT activity was measured both without and with 50 mg of pyridoxal phosphate addition to the incubation mixture. Glucocorticoid levels in plasma increased by 11% whereas in erythrocytes the increase was 55% after hypoxia. Percent stimulation of erythrocyte GOT activity with pyridoxal phosphate before exposure to hypoxia was 180% but increased to 321% after exposure. In the second set of experiments another 10 rabbits were used. First they were exposed to hypoxia without pyridoxine hydrochloride feeding and then after 7 days with 3 mg of pyridoxine hydrochloride feeding. For glucose tolerance tests the animals were fed with 1 g of glucose immediately after the hypoxic exposures. Plasma reduced glutathione (GSH), LDH and ICDH activities increased and GOT activity was depressed after hypoxic stress, but when the animals were fed pyridoxine hydrochloride prior to the exposure the enzyme activities remained unaltered after hypoxic stress. Pyridoxine hydrochloride did not alter the pattern of glucose tolerance after hypoxic stress.     

Toxicological Assessment of Arsenic-Induced Hematological Alterations and Chromosomal Aberrations in Tilapia Oreochromis mossambicus

Arsenic is an environmental contaminant and potential carcinogen. Toxicological assessment of As, which causes hematological alterations and chromosomal aberrations, was studied in freshwater fish Oreochromis mossambicus. Fish were exposed to 3 ppm, 28 ppm, and 56 ppm concentrations of sodium arsenite (NaAsO₂) and blood samples were collected after 48 h, 96 h, and 192 h of exposure. Hematological assay of exposed fish revealed abnormal mature and immature erythrocytes, deformed erythrocytes (spindle-shaped and triangular erythrocytes) and erythrocytes with segmented nuclei in all treatments. Arsenic exposure induced chromosomal aberration in a concentration-dependent manner, whereas, a decreasing trend was found after 192 h exposure. Observations on blood cells of exposed fish revealed chromosome breaks, chromatid breaks, and chromatid gaps. The alterations and aberrations of these parameters can be effectively used to assess toxicological effects of As on fish in the aquatic environment and at the same time this study elucidates the potential risks to humans who live in arsenic-contaminated areas.     

Impact of hypercholesterolemia on in vitro toxicity of N-nitrosodiethylamine: effect on lipidperoxidation of blood and tissue

In vitro treatment of erythrocytes of normal and hypercholesterolemic rats with N-nitrosodiethylamine (NDEA), an important carcinogen frequently present in human environment and food chain resulted in a marginal increase in osmotic fragility of erythrocytes without affecting their antioxygenic potential as evidenced by insignificant effect on lipid peroxidation (LPO). However, (LPO) of all the tissues (heart, lung, liver, kidney and spleen) increased significantly on in vitro treatment with NDEA. The effects were different in different tissues under different dietary conditions