Ascorbic acid (vitamin C) modulates the mutagenic effects produced by an alkylating agent in vivo

Recent reports suggest that ascorbic acid (vitamin C) inhibits tumorigenesis as well as exerts a protective effect against mutagenesis in vitro; however, there is no information on its ability to affect gene mutations induced in vivo. In this study, we have investigated the antimutagenic effects of ascorbic acid on the frequency of 6-thioguanine-resistant (6-TGr) T-lymphocytes produced in Fischer 344 rats dosed with the direct-acting alkylating agent, N-ethyl-N-nitrosourea (ENU). The freqeuncy of 6-TG^r T-lymphocytes from the spleen measured five weeks after ENU treatment indicated that ENU produced a substantial mutagenic response. Pretreatment and/or post-treatment of rats with ascorbic acid administered in the drinking water appeared to inhibit the response, but the inhibition was statistically significant only when data from the various dosing schedules were pooled. In addition, there was no clear dose-dependency to the inhibitory effect of ascorbic acid. To further evaluate the time effects of the vitamin supplement on ENU mutagenicity, rats were exposed to the mutagen together with ascorbic acid, which was given continuously for the entire duration of the experiment. At specific times after ENU treatment, the frequency of 6-TG^r T-cells was determined in lymphocytes isolated from the spleen and the thymus. Time-dependent increases in the frequency of 6-TG^r T-cells were observed with ENU treatment; ascorbic acid significantly reduced the ENU-mediated mutagenic responses, most dramatically in the spleen at weeks 6 and 8 (P < 0.0001), and to a lesser extent in the thymus (P < 0.01 at week 6 and P < 0.006 at week 8). Our data suggest that ascorbic acid intake affects the in vivo mutagenicity of ENU, a direct-acting mutagen/carcinogen, and that the reported inhibitory effects of the antioxidant on carcinogenesis may be partially mediated by its effects on mutagenesis. Although it is difficult to extrapolate from rodent studies to humans, the results presented suggest an explanation for epidemiological data that link vitamin C ingestion with decreased cancer risk. © 1994 Wiley-Liss, Inc.     

体内彗星实验联合微核实验检测化合物的遗传毒性

目的:考察体内彗星实验联合微核实验检测化合物遗传毒性的可行性。方法:以阳性化合物N-乙基-N-亚硝基脲为模型药物,取♂大鼠随机分为空白对照组和给药组[40 mg/(kg·d)],每组5只,灌胃给药3次,末次给药后3 h处死大鼠。进行彗星实验,收集大鼠外周血、肝、胃、睾丸组织经前处理制备成单细胞凝胶玻片进行电泳,使用Komet 6.0软件分析单细胞显微图像,每种组织分析100个细胞(胃组织50个细胞),计算尾部DNA百分含量。进行微核实验,收集大鼠骨髓细胞进行涂片,计数2 000个嗜多染红细胞(PCE)中含微核细胞(MNPCE)的数目及嗜多染红细胞微核率。结果:与空白对照组比较,模型药物可使大鼠外周血淋巴、肝、胃、睾丸细胞组织的尾部DNA百分含量明显增加(P<0.05),并可使大鼠骨髓嗜多染红细胞微核率明显升高(P<0.05)。结论:彗星实验可用于动物体内多种脏器遗传毒性的检测;微核实验可用于动物骨髓细胞的遗传毒性检测。     

乙基亚硝基脲致大鼠脑星形胶质细胞DNA损伤及对P53和P21蛋白表达的影响

目的研究乙基亚硝基脲(N-ethyl-N-nitrosourea,ENU)诱发原代大鼠脑星形神经胶质细胞DNA损伤及对P53、P21蛋白表达的影响.方法采用机械分离技术,在体外培养大鼠脑神经胶质细胞,通过传代培养的方法纯化大鼠脑星形胶质细胞.给予不同浓度ENU(1.7、3.4、6.8、13.6、27.2 mmol/L)染毒,采用单细胞凝胶电泳(single-cell gel electrophoresis,SCGE)和免疫组织化学的方法观察ENU对星形胶质细胞DNA的损伤作用和抑癌基因p53、p21的产物P53、P21蛋白在细胞中表达.结果不同染毒浓度下,各染毒组对星形胶质细胞均有不同程度DNA损伤作用,各染毒组拖尾细胞百分率与阴性对照组比较,差异均有统计学意义(P＜0.01),且随受试物浓度增加而增加,表现出明显的剂量-效应关系(r=0.916).各染毒组细胞DNA的迁移距离(尾长)与阴性对照组比较,差异均有统计学意义(P＜0.01).在13.6、27.2 mmol/L ENU染毒组,P53有阳性表达,与阴性对照组比较,有统计学意义(P＜0.01),而P21在各实验组均未见阳性表达.结论ENU对大鼠脑星型胶质细胞的毒性主要表现在诱导细胞遗传物质DNA损伤以及抑癌基因p53的表达水平升高.     

N－乙基－N－亚硝基脲诱发大鼠胶质瘤的实验研究

采用人工合成的Ｎ－乙基－Ｎ－亚硝基脲（ＥＮＵ，６０ｍｇ／ｋｇ体重），经胎盘和皮下给药分别在妊娠晚期孕鼠子代和３天龄Ｗｉｓｔａｒ大鼠经１２个月诱发出神经系统胶质瘤．成瘤率分别为７３．２％和６８．３％（胶质瘤为６５．９％和６３．４％）；胶质瘤以少突－星形细胞瘤和少突胶质细胞瘤为主，呈小灶性、多灶性、混合性，并伴有灶性和／或弥漫性胶质增生存在．结果表明，这两种胶质瘤模型为深入研究胶质瘤的发生、生长及分化提供了良好的途径．本文讨论了灶性胶质母细胞增生在胶质瘤发生学上的意义．     

New insights into tetrahydrobiopterin pharmacodynamics from Pah, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH₄) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah^enu1/2 bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH₄ treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH₄ pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using ¹³C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah^enu1/1, and Pah^enu1/2 mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah^enu1/1 and Pah^enu1/2 indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH₄. In conclusion, our findings show a significant impact of the genotype on the response to BH₄ in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.     

Mutational response at the splenic T-Lymphocyte hprt locus in mice treated as neonates: Contrasting effects of the carcinogens N-ethyl-N-nitrosourea,dimethylnitrosamine, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

The newborn mouse tumorigenicity assay, which involves the treatment of animals during the first two weeks after birth and monitoring tumor induction after a year, has been suggested as a cost- and time-effective alternative to the conventional two-year rodent bioassay. In order to evaluate whether or not lymphocyte hprt mutant induction is an accurate predictor of carcinogenicity in the assay, we determined the frequencies of 6-thioguanine-resistant (TG^r) lymphocytes in the spleens of mice neonatally treated with the carcinogenic mutagens N-ethyl-N-nitrosourea (ENU), dimethylnitrosamine (DMN),and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Male C57BL/6 pups were injected on post-natal days 8 and 15, and the frequency of TG^r T-lymphocytes was measured in groups of three animals, sacrificed periodically up to 31 weeks post-treatment. Compared to background frequencies of 1.1–2.9 × 10⁻⁶, mutant frequencies (MFS) reached 155.1 × 10⁻⁶ following a cumulative dose of 49 mg ENU/kg body weight and 172.3 × 10⁻⁶ following a cumulative dose of 142 mg ENU/kg. These results show that TG^r lymphocyte mutations can be induced and measured in mice treated as neonates and that the induced MFs found for mice treated neonatally with ENU are comparable with frequencies reported for the treatment of adult animals with the same chemical. In contrast, treatment with the promutagenic and procarcinogenic compounds DMN (at a maximum concentration of 10.5 mg/kg) and PhIP (26.2 mg/kg) did not result in an increase in lymphocyte MF, suggesting that reactive metabolites of these compounds may not be reaching cells that are sensitive for mutation fixation. The results indicate that the lymphocyte hprt assay may fail to predict the carcinogenicity of some test chemicals in the neonatal mouse bioassay. Environ. Mol. Mutagen. 31:243–247, 1998 © 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Identification of Mouse Cytomegalovirus Resistance Loci by ENU Mutagenesis

Host resistance to infection depends on the efficiency with which innate immune responses keep the infectious agent in check. Innate immunity encompasses components with sensing, signaling and effector properties. These elements with non-redundant functions are encoded by a set of host genes, the resistome. Here, we review our findings concerning the resistome. We have screened randomly mutagenized mice for susceptibility to a natural opportunistic pathogen, the mouse cytomegalovirus. We found that some genes with initially no obvious functions in innate immunity may be critical for host survival to infections, falling into a newly defined category of genes of the resistome.     

Sensitivity of the ΦX174 am3 allele in relation to the endogenous Hprt gene for detecting mutation in transgenic mice

Transgenic mice have been developed containing multiple, chromosomally integrated copies of the ΦX174 am3 allele that serve as reporters for in vivo mutation at a single A:T basepair. In this study, we examined the relative sensitivity of the am3 transgene for detecting the in vivo mutagenicity of N-ethyl-N-nitrosourea (ENU). Three-week-old male ΦX174 mice were treated with 0, 40, and 160 mg/kg of ENU. After 1, 3, 6, and 9 weeks, animals were killed, their spleens removed, and isolated splenocytes were used to measure mutant frequencies (MFs) in both the am3 allele and the endogenous Hprt gene. For animals treated with 40 mg/kg of ENU, the Hprt assay detected an average 22-fold increase over background, while the am3 MFs averaged threefold above background. With the 160 mg/kg dose, the Hprt assay detected a 54-fold average increase, while a sixfold average increase above background was found for the transgenic locus. We conclude that the sensitivity of the am3 assay to ENU was compromised by the presence of ex vivo mutations. Adjustment of am3 MFs to exclude these ex vivo mutants could enhance the sensitivity of the assay. Environ. Mol. Mutagen. 32:229–235, 1998 © 1998 Wiley-Liss, Inc.     

An experimental study on N-ethyl-N-nitrosourea-induced rat brain gliomas

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Magnetic Resonance Imaging of Ethyl-nitrosourea-induced Rat Gliomas: A Model for Experimental Therapeutics of Low-grade Gliomas

Human low-grade gliomas represent a population of brain tumors that remain a therapeutic challenge. Preclinical evaluation of agents, to test their preventive or therapeutic efficacy in these tumors, requires the use of animal nobreak models. Spontaneous gliomas develop in models of chemically induced carcinogenesis, such as in the transplacental N-ethyl-N-nitrosourea (ENU) rat model. However, without the ability to detect initial tumor formation, multiplicity or to measure growth rates, it is difficult to test compounds for their interventional or preventional capabilities. In this study Fisher-334 rats, treated transplacentally with ENU, underwent magnetic resonance imaging (MRI) examination in order to evaluate this approach for detection of tumor formation and growth. ENU-induced intracranial cerebral tumors were first observable in T2-weighted images beginning at 4 months of age and grew with a mean doubling time of 0.487 ± 0.112 months. These tumors were found histologically to be predominately mixed gliomas. Two therapeutic interventions were evaluated using MRI, vitamin A (all-trans retinol palmitate, RP), as a chemopreventative agent and the anti-angiogenic drug SU-5416. RP was found to significantly delay the time to first tumor observation by one month (P = 0.05). No differences in rates of tumor formation or growth rates were observed between control and RP-treated groups. MRI studies of rats treated with SU-5416 resulted in reduction in tumor growth rates compared to matched controls. These results show that MRI can be used to provide novel information relating to the therapeutic efficacy of agents against the ENU-induced tumor model.