Effects of pretreatment with pyrazole and inducers of mixed function oxidases on DNA repair elicited by dimethylnitrosamine in rat hepatocytes in vivo and in vitro

Experiments were performed to investigate the relationship between the rate of oxidative metabolism of dimethylnitrosamine (DMN) by rat liver microsomes (i.e., DMN demethylase activity, DMNd) and its genotoxicity in liver, as assessed by the in vitro and in vivo/in vitro rat hepatocyte primary culture/DNA repair (HPC/DR) assays. Pretreatment of rats with pyrazole (PYR) resulted in a 4-fold increase in DMNd and a 3-fold greater DNA repair response to in vivo administration of 5 mg DMN/kg body weight. Pretreatment with phenobarbital (PB), dichlorodiphenyltrichloroethane (DDT), 3-methylcholanthrene (3-MC), beta-naphthoflavone (beta-NF) or Aroclor 1254 (ARO) produced a variable degree of inhibition of DMNd and had no significant effects on the response to DMN in the in vivo/in vitro HPC/DR assay. DNA repair elicited by DMN in vitro was decreased in hepatocytes from rats pretreated with 3-MC, while PB, DDT, beta-NF and ARO pretreatments had little effect on the response. In contrast, PYR pretreatment produced a 4.5-6.7-fold increase in the in vitro DNA repair response to DMN, and extended detection of positive responses to lower concentrations. Most of the inducers had no effect on DNA repair elicited by the direct acting alkylator, methyl methanesulfonate (MMS). Thus, the pretreatment-related changes in DMN-induced DNA repair were probably due to alterations in DMNd rather than to effects on the DNA repair capacity of the hepatocytes.     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

新生大鼠低氧缺血性脑损伤对脑组织病理形态和神经营养因子的影响

目的改进新生大鼠低氧缺血性脑损伤（HIBI）模型的制作方法,观察低氧缺血对脑组织病理形态和神经营养因子的影响。方法大鼠随机分为空白对照组（n=5）、假手术组（n=8）和HIBI模型组（n=19）,HIBI模型制作中省去了经典Rice法中的麻醉步骤和动物手术后休息时间,观察HIBI后大鼠体重增长情况,行为能力表现以及脑组织病理形态学改变;比较HIBI制模后3 d假手术组及HIBI组鼠脑匀浆beta-NGF和human-NT3的变化。结果 （1）HIBI模型组体重增长明显落后于空白对照组和假手术组（P〈0.01）;（2）HIBI组全部出现不同程度的行为异常：84%翻身不能,63%肌肉颤动和/或头颤,抽搐者占42%,死亡率为21%。制模后3 d HIBI模型组大鼠的行为障碍和异常运动的发生率均明显低于制模当日（P〈0.01）;（3）HE染色可见HIBI模型组大鼠左侧大脑半球神经元损伤及神经胶质细胞增生;（4）制模后3 d鼠脑匀浆human-NT3含量较假手术组增加（P〈0.05）;β-NGF含量无明显变化。结论制作的新生大鼠HIBI模型更符合临床新生儿HIBI的自然病程。HIBI早期神经营养因子表达增加在神经保护机制中发挥重要作用。     

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test

Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec⁻) double mutant mei-9^a mei-41^D5 males and Rec⁺ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level, the experiment was repeated with a larval stock consisting of Rec⁺ males and Rec⁺ females under comparable conditions. A preferential killing effect upon Rec⁻ larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor

Summary Induction of hepatocyte DNA synthesis in culture by cyproterone acetate (CPA), a potent hepatomitogen in vivo, was studied. Adult rat hepatocytes were grown on collagen gels in primary culture for 3 to 10 d. Epidermal growth factor (EGF) was used as a model inducer to establish appropriate culture conditions. (a) In serum-free medium EGF stimulated a wave of DNA synthesis in 10 to 30% of the hepatocytes. CPA had only a weak effect. (b) Increasing concentrations of newborn bovine serum (NBS) at 5 to 95% progressively inhibited the stimulatory effect of EGF. A similar inhibition was obtained by adding bovine serum albumin; 20% NBS, however, had a slightly stimulatory effect on the induction of DNA synthesis by CPA. (c) Portal rat serum (RS) at concentration of 5 to 95% markedly stimulated DNA synthesis, a plateau being reached between 20 and 95%. EGF had a distinct enhancing effect on DNA synthesis in the presence of 5 and 20% RS but not at 50 and 95%. CPA stimulated DNA synthesis in the presence of 20, 50, and 95% RS in a synergistic way. (d) Mitoses were found after treatment with EGF or with CPA. These results show that CPA can induce DNA synthesis in cultured hepatocytes and that RS contains factors facilitating the response to CPA. This study was supported by Gesellschaft für Strahlen-und Umweltforschung mbH, München, Germany.     

Synthesis of barbituric acid derivatives and their effect on survival of functional hepatocytes from adult rats in primary culture

Summary Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture. Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture. Especially C-5, 6, and 9 showed excellent efficiency for such action. The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation. The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media. Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes. The addition of dexamethasone (10 μM) caused a 1.7 to 2.1-fold induction in TAT activity. The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.     

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: Comparison with quercetin

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells’ capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.     

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl

BACKGROUND AND AIMS: The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. METHODS: Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. KEY RESULTS: Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. CONCLUSIONS: Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.     

Effects of postnatal treatment with naloxone on plasma gonadotropin,prolactin, testosterone and testicular functions in male rats

Opioid peptides are implicated in the control of gonadotropin and prolactin secretion. The role of opioid antagonist naloxone and its effects on plasma gonadotropin, prolactin, testosterone levels and testicular hyaluronidase, acid phosphatase, [³H]uridine and thymidine incorporation, RNA, DNA and protein concentrations were evaluated in rats after administration of naloxone beginning day 1 through 21 and autopsied on 45, 60 and 90 days of age. Plasma gonadotropin and testosterone levels were significantly elevated after naloxone treatment. Testicular hyaluronidase and acid phosphatase activity increased till 60 days post treatment and declined thereafter. Concentrations of RNA and protein did not change significantly but the concentration of DNA declined at 45 and 60 days of age. These results suggest that endogenous opioid peptides exert regulatory influence on gonadotropin secretion which in turn control the testicular function in the male rat.     

Effects of physical exercise training in DNA damage and repair activity in humans with different genetic polymorphisms of hOGG1 (Ser326Cys)

The main purpose of this pilot study was to investigate the possible influence of genetic polymorphisms of the hOGG1 (Ser326Cys) gene in DNA damage and repair activity by 8‐oxoguanine DNA glycosylase 1 (OGG1 enzyme) in response to 16 weeks of combined physical exercise training. Thirty‐two healthy Caucasian men (40–74 years old) were enrolled in this study. All the subjects were submitted to a training of 16 weeks of combined physical exercise. The subjects with Ser/Ser genotype were considered as wild‐type group (WTG), and Ser/Cys and Cys/Cys genotype were analysed together as mutant group (MG). We used comet assay in conjunction with formamidopyrimidine DNA glycoslyase (FPG) to analyse both strand breaks and FPG‐sensitive sites. DNA repair activity were also analysed with the comet assay technique. Our results showed no differences between DNA damage (both strand breaks and FPG‐sensitive sites) and repair activity (OGG1) between genotype groups (in the pre‐training condition). Regarding the possible influence of genotype in the response to 16 weeks of physical exercise training, the results revealed a decrease in DNA strand breaks in both groups, a decrease in FPG‐sensitive sites and an increase in total antioxidant capacity in the WTG, but no changes were found in MG. No significant changes in DNA repair activity was observed in both genotype groups with physical exercise training. This preliminary study suggests the possibility of different responses in DNA damage to the physical exercise training, considering the hOGG1 Ser326Cys polymorphism. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of Pectin from the Seagrass Zostera marina on the Content of DNA and RNA in Rat Hepatocytes Contaminated by Lead

The influence of zosterin-pectin of the seagrass Zostera marina on the content of DNA and RNA in rat hepatocytes under normal conditions and with lead contamination has been studied. It was shown that zosterin increases the share of the high-ploidy (8c) nuclei and the content of the nuclear RNA of the intact animals and, against a background of toxicant action, normalizes the ratio of the ploidy classes and further increases the RNA mass.     

Hepatoprotective effect of total flavonoids from Laggera alata against carbon tetrachloride-induced injury in primary cultured neonatal rat hepatocytes and in rats with hepatic damage

Summary The hepatoprotective activities of total flavonoids of Laggera alata (TFLA) were evaluated by carbon tetrachloride (CCl₄)-induced injury in primary cultured neonatal rat hepatocytes and in rats with hepatic damage. In vitro, TFLA at a concentration range of 1–100 g/ml improved cell viability and inhibited cellular leakage of two enzymes, hepatocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT), caused by CCl₄. In vivo, oral treatment with TFLA at doses of 50, 100, and 200 mg/kg significantly reduced the levels of AST, ALT, total protein, and albumin in serum and the hydroxyproline and sialic acid levels in liver. Histopathological examinations revealed that liver damage were improved when treated with TFLA. Meanwhile, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals scavenging activities of TFLA were also determinated. To understand the exact components of TFLA responsible for the hepatoprotective effect, nine flavonoid compounds were isolated and identified from TFLA. In conclusion, the present investigation was the first to verify the hepatoprotective effect of L. alata in vitro and in vivo. The hepatoprotective action of TFLA is likely related to its potent antioxidative and anti-inflammatory activity. Neutralizing reactive oxygen species by nonenzymatic mechanisms and enhancing the activity of original natural hepatic-antioxidant enzymes may be the main mechanisms of TFLA against CCl₄-induced injury.     

Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats. The use of 14C-labelled lactate in studies of hepatic gluconeogenesis

1. Hepatocytes from starved rats were incubated with l-lactate and NH(4)Cl or norvaline, and the rates of the tricarboxylic acid cycle and of gluconeogenesis were calculated from changes in metabolite concentrations or from radioisotopic data from incubations with labelled lactate or propionate. 2. Gluconeogenesis was stimulated by the addition of 10mm-NH(4)Cl, 5mm-norvaline or 1mm-oleate by 27, 45 and 59% respectively. NH(4)Cl or norvaline also increased lactate uptake. Norvaline inhibited urea synthesis from NH(4)Cl by 85%. 3. The effects of NH(4)Cl and norvaline were not additive. However, NH(4)Cl inhibited and norvaline was without effect on gluconeogenesis from pyruvate, indicating that the two compounds act by different mechanisms. 4. The tricarboxylic acid-cycle flux was increased 80% by lactate, and NH(4)Cl caused a further 25% stimulation. Norvaline had no effect on the tricarboxylic acid-cycle flux. NH(4)Cl and norvaline tripled and doubled, respectively, flux through pyruvate dehydrogenase. 5. Total ATP formation was calculated to range from 470 to 830mumol/h per 100mg of protein, of which the basic metabolic activity accounted for 400-450mumol/h per 100mg of protein. ATP formation does not seem to be rate-limiting for gluconeogenesis. 6. Pyruvate recycling was estimated from the (14)C yield from [1-(14)C]propionate in lactate and glucose to be 10-30% of the flux of phosphoenolpyruvate to glucose. The further addition of NH(4)Cl more than doubled the recycling of pyruvate. 7. [1,4-(14)C]Succinate was rapidly metabolized by hepatocytes. About 20% of the radioactivity was recovered in glucose, indicating that succinate is also metabolized by intact (non-damaged) hepatocytes. 8. It is concluded that the metabolism of lactate by the liver is too complex to allow simple rate measurements with labelled compounds.     

血必净注射液对缺氧/复氧大鼠心肌功能与结构的影响

目的：探讨不同剂量的血必净注射液对缺氧/复氧大鼠心肌功能的保护作用。方法：采用Langendorff方法制备大鼠离体心脏缺氧/复氧模型。130只雄性SD大鼠随机分为对照组（sham组）,缺氧/复氧组（H/R组）,低、中、高剂量血必净组（XBJ_L、XBJ_M、XBJ_H组）,除对照组外,其他四组按复氧不同时相（复氧0.5 h、1 h、2 h）又分别分为3个亚组（n=10）。对照组在平衡灌注20 min时纪录左室发展压（LVDP）、左心室发展压最大上升/下降速率（±dp/dt_max）、左心室内压（LVP）、心率（HR）的值, ELISA检测心肌中肌酸激酶同工酶（CK-MB）的浓度,光镜下观察心肌组织结构的改变;其余各组平衡灌注20 min后,灌注ThomasⅡ停搏液使心脏完全停搏30 min之后复灌K-H液使其心脏复跳,连续记录LVDP、±dp/dt_max、LVP、HR在复氧不同时间点的动态变化,ELISA检测各组复氧不同时间点心肌中CK-MB的浓度,光镜下观察各组复氧不同时间点心肌组织结构的改变。结果：与sham组相比,其余各组LVDP、±dp/dt_max、LVP值均降低（P<0.05）,心肌中CK-MB浓度上升（P<0.05）,心肌组织结构发生异常改变,随着复氧时间延长,以上指标异常变化逐渐加剧;在复氧0.5 h、1 h、2 h,各剂量血必净组LVDP、±dp/dt_max、LVP的值均高于H/R组对应时间点的值（P<0.05）,心肌中CK-MB浓度均低于H/R组,心肌组织结构异常变化减轻,以中剂量改善效果最佳（P<0.05）。结论：血必净注射液能够有效改善缺氧/复氧大鼠心肌的功能及形态学结构,以中剂量血必净（4 ml/100 ml）效果最佳。     

Ameliorative effect of supplementation with l-glutamine on oxidative stress,DNA damage,cell viability and hepatotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat hepatocyte cultures

The most potent of the dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a persistent and ubiquitous environmental contaminant. And the health impact of exposure to TCDD is of great concern to the general public. Recent data indicate that l-glutamine (Gln) has antioxidant properties and may influence hepatotoxicity. The objective of the present study was undertaken to explore the effectiveness of Gln in alleviating the hepatotoxicity of TCDD on primary cultured rat hepatocytes. Gln (0.5, 1 and 2 mM) was added to cultures alone or simultaneously with TCDD (0.005 and 0.01 mM). The hepatocytes were treated with TCDD and Gln for 48 h. Then cell viability was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and lactate dehydrogenase (LDH) release, while total antioxidant capacity (TAC), total glutathione (TGSH) and total oxidative stress (TOS) levels were determined to evaluate the oxidative injury. The DNA damage was also analyzed by liver micronucleus assay (MN) and 8-oxo-2-deoxyguanosine (8-OH-dG). The results of MTT and LDH assays showed that TCDD decreased cell viability but not l-glutamine. TCDD also increased TOS level in rat hepatocytes and significantly decreased TAC and TGSH levels. On the basis of increasing doses, the dioxin in a dose-dependent manner caused significant increases of micronucleated hepatocytes (MNHEPs) and 8-OH-dG as compared to control culture. Whereas, in cultures exposured with Gln alone, TOS levels were not changed and TAC and TGSH together were significantly increased in dose-dependent fashion. The presence of Gln with TCDD modulated the hepatotoxic effects of TCDD on primary hepatocytes cultures. Noteworthy, Gln has a protective effect against TCDD-mediated DNA damages. As conclusion, we reported here an increased potential therapeutic significance of l-glutamine in TCDD-mediated hepatic injury for the first time.