Metabolism of dimethylnitrosamine to mutagenic intermediates by kidney microsomal enzymes and correlation with reported host susceptibility to kidney tumors.

Purified kidney microsomal enzymes from C3H/HeJ and Ha/1CR mice exhibited an unusually high capacity to generate mutagenic metabolites from dimethylnitrosamine (DMNA) when compared with similar enzyme preparations from BALB/cJ and RF/J mice. These results suggested that the DMNA-activating enzymes involved in mutagen formation were either present at higher levels or were more active in the kidneys of C3H/HeJ and Ha/ICR mice than in those of the other two strains. This strain difference correlated with the established susceptibility of these four strains to the neoplastic activity of DMNA.     

A liver DNA synthesis promoter induced in rat plasma by injection of dimethylnitrosamine (DMNA) or thioacetamide

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.     

Effect of phenobarbital on diethylnitrosamine and dimethylnitrosamine induced hepatocellular tumors in male B6C3F1 mice

The effect of the type of carcinogen initiator on the ability of phenobarbital (PB) to promote hepatic tumor formation in 15-day-old initiated male B6C3F1 mice was evaluated. Fifteen-day-old male B6C3F1 mice were divided into 6 groups of 10 mice each. Groups 1 and 2 received a single intraperitoneal (i.p.) injection of diethylnitrosamine (DENA) (5 micrograms/body wt). Groups 3 and 4 received a single i.p. injection of diethylnitrosamine (DENA) (5 micrograms/g body wt). Groups 3 and 4 received a single i.p. injection of dimethylnitrosamine (DMNA) (5 micrograms/g body wt). Groups 5 and 6 received a single i.p. injection of saline. At weaning (28 days of age), mice in groups 2, 4 and 6 received PB (500 mg/ml) in their drinking water. Mice in groups 1, 3 and 5 received deionized drinking water. Drinking water treatment continued for 24 weeks at which time mice were sampled. At sampling, mice were examined for hepatic tumors by histology. Mice in groups 5 (no treatment) and 6 (PB only) did not exhibit hepatic tumors. Groups 2 (DENA + PB) displayed a decrease in hepatic adenomas from that of group 1 (DENA only), confirming previous observations. Treatment with DMNA and PB (group 4), however, resulted in a significant increase in both hepatic adenoma incidence and number over that of DMNA only (group 3) treated mice. The promoted adenomas appeared to be predominantly eosinophilic in appearance. The type of initiator therefore appears important in determining if 15-day-old initiated male B6C3F1 mice respond to the promotion effects of PB.     

Early effects of dimethylnitrosamine on protein chain initiation and postmicrosomal polyadenylic acid-containing RNA content in mouse liver.

A mouse liver S-30 system was used to study the early effects of dimethylnitrosamine (DMNA) on polypeptide chain initiation and messenger RNA content. The inhibition of protein synthesis after DMNA administration was associated with a reduced capacity of the S-30 system to form 80S ribosomal initiation complexes. The binding of formylatable methionyl transfer RNA to polysomes was also depressed. The initiation defect was detectable in the assay system slightly later than the decrease in protein synthesis. Addition of mRNA stimulated both translation and 80S initiation complex formation but could not fully restore the activity of the S-30 system from DMNA-treated mice. A loss of poly(A)+ RNA from the postmicrosomal subfraction of the S-30 fraction was observed as early as 15 min after DMNA administration. Later, polyriboadenylic acid also decreased in the microsomal fraction. Monosomes accumulating in response to DMNA treatment were deficient in mRNA as measured by polyriboadenylic acid analysis. Conversely, the proportion of polyriboadenylic acid in the remaining polysomes increased, indicating that the mRNA had become less densely occupied with ribosomes.     

Strain differences in hepatic tumor promotion by phenobarbital in diethylnitrosamine- and dimethylnitrosamine-initiated infant male mice

The effects of phenobarbital (PB) on hepatocellular carcinogenesisin three strains of nitrosamine-initiated infant male mice wereevaluated. Fifteen-day-old C57Bl/6NCr1BR (C57Bl), C3H/HeNCr1BR(C3H) and B6C3F1 mice were treated with a single i.p. injectionof either diethylnitrosamine (DENA) (5 µg/body wt), dimethylnitrosamine(DMNA) (5 µg/body wt) or saline. One-half of the treatedmice received PB via the drinking water (500 mg/l) for 24 weeks.The remaining treated mice were given deionized drinking water.Mice were killed at 28 weeks of age and hepatic lesions wereevaluated. Only animals that received DENA or DMNA exhibitedtumors. C3H mice treated with DENA + PB demonstrated a significantincrease in hepatic adenoma number compared to C3H mice exposedto DENA only. Conversely, B6C3F1 males treated with DENA + PBexhibited a significant decrease in the number of hepatic adenomascompared to B6C3F1 males treated with DENA alone. No changewas noted in adenoma size in B6C3F1 mice treated with DENA +PB from those receiving DENA only. Chronic PB exposure of C57B1males previously treated with DENA had no effect on hepaticadenoma number or size. C3H mice treated with DMNA + PB displayedan increase in both adenoma size and adenoma number comparedto C3H mice receiving DMNA only. Similarly, in B6C3F1 mice,PB treatment increased both the adenoma incidence and adenomanumber in DMNA initiated mice. PB had no effect on hepatic adenomaincidence or number in DMNA-treated C57B1 mice. These data suggestthat the ability of PB to promote hepatic tumorigenesis in the15-day-old initiated mouse is dependent on both the strain ofthe mouse and the initiating chemical carcinogen.     

Tumor induction by N-nitroso compounds in bivalve mollusks Unio pictorum

Diethylnitrosamine (DENA) and dimethylnitrosamine (DMNA) dissolved in tank water induced neoplasms in bivalve mollusks Unio pictorum. DENA induced tumors in 18% of the animals exposed to 200 ppm and 68% of those exposed to 400 ppm; average latent periods were 71 and 61 days, respectively. DMNA induced tumors in 8% of the animals exposed to 200 ppm for 51 days and 27% of those exposed for 152 days with average latent periods of 85 and 81 days, respectively. Neoplasms were observed in the digestive gland (basophilic cell tumors; 38 cases), the hemopoietic system (hemocytoblastosis; 12 cases) and the kidney (poorly differentiated carcinoma; 1 case). It is expedient to use mollusks, both for testing of N-nitroso compounds and as a biologic indicator of hydrospheric pollution.     

DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.     

The binding of dimethylnitrosamine to nucleic acids catalyzed by liver enzymic fractions.

The in vitro binding of a metabolite of dimethylnitrosamine (DMNA) to calf thymus DNA catalyzed by a microsomal system from rat liver is reported. The amount ob binding is unaffected by using microsomes induced by 3-methylcholanthrene and/or normal or induced pH 5 enzymes. The microsomal system is also effective in catalyzing covalent binding of the nitroso compound with synthetic polyribonucleotides.     

DNA adducts formed by the comutagens harman and norharman in various tissues of mice

Covalent modifications of DNA in various tissues of mice with harman or norharman were analyzed by 32P-postlabeling assay. Administration of 0.1% harman to mice in their diet for 4 weeks resulted in DNA adducts in the liver and kidney. No specific DNA adduct was detected in other tissues, such as the glandular stomach, large intestine and brain. Similar treatment of mice with norharman resulted in DNA adducts in the kidney, glandular stomach and large intestine, but not in the liver or brain. These results suggests the in vivo genotoxicities of harman and norharman.     

Effect of N-nitroso compounds and aminoazo compound on organ cultures of human embryonal kidney]

It was shown that embryonal tissue of human kidney could be explanted into organ cultures during 3-4 weeks. In the immediate effect of N-methyl nitrosurea (NMU), dimethyl nitrosamine (DMNA) and orthoamino-azotoluole (OAAT) a considerable number of hyperplastic epithelial growths was revealed, various types of morphological changes: tubular epithelium hyperplasia, glomerular epithelium hyperplasia and mixed hyperplasia (simultaneous hyperplasia in tubular and glomerular epithelium) being noted. Their frequency increased as the experiment proceeded, and also was dependent on the carcinogen concentration in a nutrient medium. NMU mostly produced a hyperplastic growth in tubular epithelium, DMNA--in glomerular epithelium while OAAT resulted in enhancement of hyperplastic processes in tubular and glomerular epithelium.     

Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncated pseudomonas exotoxin

To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. A431-CD30 cells were injected s.c. on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v. Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated. Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies. Published 2001 Wiley-Liss, Inc.     

Effect of Adriamycin on DNA, RNA and Protein Biosyntheses in Mouse Tissues, in Connection with Its Cardiotoxicity

We examined whether the cause of the remarkable decreases in the activities of lipid peroxidationpreventive enzymes in the heart of adriamycin (ADR)-treated mice might be related to inhibition of DNA, RNA or protein biosynthesis. It was found that biosyntheses of DNA, RNA and protein in the heart, liver and kidney of mice were markedly inhibited by ADR (15 mg/kg, ip). The inhibitory effects of ADR on each type of biosynthesis were particularly marked in the heart among the tissues examined. Strong correlations between the percentage inhibition of DNA and protein biosynthesis by ADR, and the percentage decrease in the activities of lipid peroxidationpreventive enzymes were observed in the heart, liver, kidney and lung, especially for the decrease of glutathione peroxidase activity and the inhibition of DNA and protein biosyntheses. We also found that marked decreases of DNA, RNA and protein biosyntheses in ADR-treated mice occurred not only in the heart but also in tumor tissues. From these results, we conclude that the increment of cardiac lipid peroxide in ADR-treated mice, which is closely related to the cardiotoxicity of ADR, results from inhibition of DNA, RNA and protein biosyntheses after the distribution of ADR.     

Effect of adriamycin on DNA, RNA and protein biosyntheses in mouse tissues, in connection with its cardiotoxicity

We examined whether the cause of the remarkable decreases in the activities of lipid peroxidation-preventive enzymes in the heart of adriamycin (ADR)-treated mice might be related to inhibition of DNA, RNA or protein biosynthesis. It was found that biosyntheses of DNA, RNA and protein in the heart, liver and kidney of mice were markedly inhibited by ADR (15 mg/kg, ip). The inhibitory effects of ADR on each type of biosynthesis were particularly marked in the heart among the tissues examined. Strong correlations between the percentage inhibition of DNA and protein biosynthesis by ADR, and the percentage decrease in the activities of lipid peroxidation-preventive enzymes were observed in the heart, liver, kidney and lung, especially for the decrease of glutathione peroxidase activity and the inhibition of DNA and protein biosyntheses. We also found that marked decreases of DNA, RNA and protein biosynthesis in ADR-treated mice occurred not only in the heart but also in tumor tissues. From these results, we conclude that the increment of cardiac lipid peroxide in ADR-treated mice, which is closely related to the cardiotoxicity of ADR, results from inhibition of DNA, RNA and protein biosyntheses after the distribution of ADR.     

Formation and persistence of benzo[a]pyrene--DNA adducts in different tissues of C57BL/10 and DBA/2 mice

Synchronous fluorescence spectrophotometry (SFS) developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)--DNA adducts was used to measure the formation and disappearance of DNA adducts in the lung, liver, kidney, spleen and small intestine of genetically responsive C57BL/10 (B10) and nonresponsive DBA/2 (D2) mice. After single stomach intubation of 100 mg/kg of benzo[a]pyrene (B[a]P) in both strains, binding of BPDE to DNA reached a peak 48 h after treatment. However, the levels of binding in the lung, liver, kidney and spleen were higher in D2 than in B10 mice. In contrast to this, in the small intestine the higher level of BPDE binding was found in B10 mice and reached its maximum 24 h earlier. Thereafter a very rapid drop in the level of BPDE--DNA adducts to a value of approximately 50% after 48 h was observed in this tissue. In the other tissues of the B10 mice the rate of adducts removal was slower, but by 14 days after treatment 90-100% of adducts were removed. In the D2 mice up to the 4th day after treatment the rates of removal of the BPDE--DNA adducts were similar to that of the B10 mice. Thereafter the level of bound hydrocarbon decreased at a slower rate. During the whole period after B[a]P treatment distinct differences between organs in the amount of BPDE--DNA adducts were observed. In D2 mice the highest level of binding was found in the spleen followed by the lung, kidney, liver and small intestine. In B10 mice the highest level of binding was observed in the DNA of small intestine. The data suggest that the decreased rate of B[a]P metabolism in D2 mice may be at least in some tissues the reason of higher binding of BPDE--DNA adducts in comparison with B10 mice.     

3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮对小鼠的遗传毒性与氧化损伤的关系

背景与目的：研究饮水氯化消毒副产物3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮(3-chloro-4-(dichlommethyl)-5-hydroxy-2[5H]-furanone,MX)对小鼠的遗传毒性与氧化损伤的诱导。材料与方法：昆明种小鼠随机分为4组,即溶剂对照组与低、中、高3个MX剂量组(11、33、100mg／kg),受试小鼠经腹腔注射染毒3h后处死。应用单细胞凝胶电泳技术(SCGE,彗星分析)检测小鼠肝、肾、小肠的DNA损伤,并测定小鼠肝、肾和小肠中脂质过氧化主要终产物丙二醛(Malondialdehyde,MDA)的含量。结果：随着MX浓度的增加,小鼠肝、肾、小肠中MDA含量与其细胞的Olive尾矩明显升高,且呈现较好的剂量依赖性。与溶剂对照组比较：①MX各剂量组小肠细胞Olive尾距均显著性增加,肝、肾细胞的Olive尾距在MX较高剂量时有显著性增加;②中、高剂量组小鼠肝组织中MDA含量显著性升高,高剂量组小鼠肾和小肠组织中MDA含量明显升高,差异有显著性意义。肝、肾、小肠MDA含量与Olive尾矩均呈明显正相关。结论：MX可引发哺乳动物多种脏器的脂质过氧化反应增强;并能引起细胞DNA损伤。MX导致机体组织的氧化损伤可能在细胞遗传毒性作用中起重要作用。     

A novel strategy inducing autophagic cell death in Burkitt's lymphoma cells with anti-CD19-targeted liposomal rapamycin

Relapsed or refractory Burkitt''s lymphoma often has a poor prognosis in spite of intensive chemotherapy that induces apoptotic and/or necrotic death of lymphoma cells. Rapamycin (Rap) brings about autophagy, and could be another treatment. Further, anti-CD19-targeted liposomal delivery may enable Rap to kill lymphoma cells specifically. Rap was encapsulated by anionic liposome and conjugated with anti-CD19 antibody (CD19-GL-Rap) or anti-CD2 antibody (CD2-GL-Rap) as a control. A fluorescent probe Cy5.5 was also liposomized in the same way (CD19 or CD2-GL-Cy5.5) to examine the efficacy of anti-CD19-targeted liposomal delivery into CD19-positive Burkitt''s lymphoma cell line, SKW6.4. CD19-GL-Cy5.5 was more effectively uptaken into SKW6.4 cells than CD2-GL-Cy5.5 in vitro. When the cells were inoculated subcutaneously into nonobese diabetic/severe combined immunodeficiency mice, intravenously administered CD19-GL-Cy5.5 made the subcutaneous tumor fluorescent, while CD2-GL-Cy5.5 did not. Further, CD19-GL-Rap had a greater cytocidal effect on not only SKW6.4 cells but also Burkitt''s lymphoma cells derived from patients than CD2-GL-Rap in vitro. The specific toxicity of CD19-GL-Rap was cancelled by neutralizing anti-CD19 antibody. The survival period of mice treated with intravenous CD19-GL-Rap was significantly longer than that of mice treated with CD2-GL-Rap after intraperitoneal inoculation of SKW6.4 cells. Anti-CD19-targeted liposomal Rap could be a promising lymphoma cell-specific treatment inducing autophagic cell death.     

Effects of NQO1 deficiency on levels of cyclopurines and other oxidative DNA lesions in liver and kidney of young mice

I-compounds are bulky indigenous DNA adducts that can be detected by (32)P-postlabeling. A subgroup, termed type II I-compounds, represents DNA lesions induced by oxidative stress. Several major type II I-compounds have been identified as dinucleotides containing 3'-terminal 8,5'-cyclo-2'-deoxyadenosine (cA). Levels of type II I-compounds depend on the pro-oxidant status of the cell. For example, enhanced formation of such oxidative DNA lesions in newborn rodents appears to be a consequence of incomplete development of neonatal antioxidant defense systems. We tested the hypothesis that young mice deficient in NAD(P)H:quinone oxidoreductase 1 (NQO1), an antioxidant enzyme catalyzing the detoxification of quinones and their derivatives, show increased formation of these oxidative DNA lesions. Type II I-compound levels were determined by (32)P-postlabeling in liver and kidney DNA of untreated male wild-type or NQO1-null C57BL/6 mice of different ages. NQO1 catalytic activities and contents were measured by spectrophotometric and Western blotting techniques, respectively. Elevated oxidative adduct levels including those containing cA were detected in NQO1-null compared to wild-type mice at 10, 30 and 90 days in liver and at 30 and 90 days in kidney DNA. Furthermore, there were statistically significant inverse relationships between type II I-compound levels and NQO1 activities in wild-type mice up to 30 days of age. Taken together, the results suggest that NQO1 plays an important role in attenuating endogenous oxidative DNA damage in vivo. Our results show also that type II I-compounds represent useful and sensitive biomarkers with utility in studies of oxidative DNA damage and its consequences.     

An in vivo-in vitro short term carcinogenicity test using rat peritoneal cells

A new short-term mammalian carcinogenicity test system is presented in which an in vivo application in rats is followed after 2 weeks by an in vitro culture of the peritoneal cells. One week of culture was sufficient to induce colony growth in soft agar in some cultures of the dimethylnitrosamine (DMNA)-treated, but not in those of control, rats. The results proved to be reproducible and dose-related. Suspended colony growth was not observed in cultures of control rats stimulated successively by the 2 mitogens: thioglycollate and Al(OH)₃. The incidence of rats showing transformed colonies in their cultures after a single DMNA dose of 20 mg/kg was nearly double that reported for rats developing tumors after a higher single dose of 30 mg/kg DMNA. More recent experiments have shown that whereas the carcinogen benzo[a]pyrene was positive, its non-carcinogenic analog benzo[e]pyrene was negative; both applied at a single oral dose of 15 mg/kg. A similar result was observed with the acetylaminofluorene (AAF) analogs: 2-AAF and 4-AAF, where the former, a carcinogen, proved positive and the latter, a non-carcinogen, negative. Both analogs were applied at a single oral dose of 50 mg/kg. 2-AAF was also positive at a dose of 25 mg/kg corresponding to 2.5% of the highest tolerable dose of this carcinogen. These results suggest a high structure-related specificity of our test system. Many more similar tests with carcinogen/non-carcinogen analogs will be needed however, before this specificity can be sufficiently established.     

High accumulation of oxidative DNA damage, 8-hydroxyguanine,in Mmh/Ogg1 deficient mice by chronic oxidative stress

8-Hydroxyguanine (8-OH-G) is a major pre-mutagenic lesion generated from reactive oxygen species. The Mmh/Ogg1 gene product plays a major role in maintaining genetic integrity by removing 8-OH-G by way of the base excision repair pathway. To investigate how oxidative stress influences the formation of 8-OH-G in Ogg1 mutant mice, a known oxidative agent, potassium bromate (KBrO(3)), was administered at a dose of 2 g/l in the drinking water to Ogg1(+/+), Ogg1(+/-) and Ogg1(-/-) mice for 12 weeks. Apurinic (AP) site lyase activity, measured by the excision of 8-OH-G from synthetic oligonucleotides, remained unchanged in kidney cell extracts isolated from Ogg1 mutant mice when the mice were pre-treated by KBrO(3). The levels of 8-OH-G in kidney DNA tremendously increased in a time-dependent manner following exposure of Ogg1(-/-) mice to KBrO(3). Of particular note, the amount of 8-OH-G in kidney DNA from Ogg1(-/-) mice treated with KBrO(3) was approximately 70 times that of KBrO(3)-treated Ogg1(+/+) mice. The accumulated 8-OH-G did not decrease 4 weeks after discontinuing treatment with KBrO(3). KBrO(3) treatment for 12 weeks gave rise to increased mutation frequencies at the transgenic gpt gene in Ogg1(+/+) mice kidney. Absence of the Ogg1 gene further enhanced the mutation frequency. Sequence data obtained from gpt mutants showed that the accumulated 8-OH-G caused mainly GC-->TA transversion and deletion. Other mutations including GC-->AT transition also showed a tendency to increase. These results indicate that 8-OH-G, produced by chronic exposure to exogenous oxidative stress agents, is not repaired to any significant extent within the overall genome of Ogg1(-/-) mice kidney.