Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N′-nitro-N-nitrosoguanidine

Outbred 7-week old male Wistar rats were exposed for 21 days to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water and N7-methyl deoxyguanosine 3'-monophosphate (N7-MedGp) levels in DNA from the pyloric mucosa (target tissue) and white blood cells (wbc: non-target tissue) were determined by ³²P-postlabelling. Exposure to MNNG resulted in the non-linear, dose-related formation of N7-medGp in both tissues. Adduct levels in the pyloric mucosa were determined to be 1058, 5.4 and 1.1 μmole N7-medGp mole^-1 deoxyguanosine 3'-monophosphate (dGp) after exposure to 4.1, 0.62 and 0.006 mg MNNG kg^-1 day^-1 respectively whereas adduct levels in the wbc DNA were lower at 5.2, 0.52 and 0.68 μmoles N7-medGp mole^-1 dGp after exposure to 4.1, 0.62 and 0.062 mg MNNG kg^-1 day^-1 respectively. In addition, the persistence of N7-medGp was investigated. Loss of adduct occurred rapidly, with a decrease of 87 and 97% respectively in target tissue and wbc DNA by 48 h after cessation of 4.1 mg MNNG kg^-1 day^-1 exposure; 14 days post-MNNG treatment, however, N7-medGp was still detectable (0.46 μmole N7-medGp mole^-1 dGp) in pyloric mucosal DNA. The quantitation of N7-medGp after exposure to low doses of carcinogen, i.e. 0.006 mg MNNG kg^-1 day^-1, approaching environmentally relevant levels has not been previously reported, and indicates that the ³²P-postlabelling assay developed here possesses sufficient sensitivity to quantitate N7- medGp in human DNA arising from environmental exposure to methylating agents.     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^-1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^-1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^-1 hair) and FB 2 (mean 11.1 μg kg^-1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Associations between smoking, GST genotypes and N7-methylguanine levels in DNA extracted from bronchial lavage cells

N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99 +/-20.3)x10(-7) versus (0.58+/-0.50)x10(-7) N7-MedGp/deoxyguanosine-3'-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59+/-15.6)x10(-7) N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1+/-38.0)x10(-7) versus (2.73+/-4.07)x10(-7) N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5+/-13.1)x10(-7) versus (2.64+/-3.89)x10(-7) N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Stoichiometric characteristics of carbon,nitrogen and phosphorus of Sibiraea angustata shrub on the eastern Qinghai-Xizang Plateau

Aims Little is known about the stoichiometric characteristics of carbon (C), nitrogen (N) and phosphorus (P) in plateau shrubs across China. Sibiraea angustata is a typical and representative shrub species on the eastern Qinghai- Xizang Plateau, and exploring its C, N and P distribution patterns and stoichiometric properties in different organs (including root, shoot, leaf, twig and fruit) would help us better understand the mechanisms of C, N and P cycling and balance in the S. angustata dominated shrub ecosystem.
Methods Sixteen sampling sites were selected on the eastern Qinghai-Xizang Plateau by the stratified sampling method. The height and coverage of the dominant shrubs, latitude, longitude and altitude of the sites were recorded. Three 5 m × 5 m plots were selected at each site. At least 128 biological samples of plant organs of S. angustata were collected and measured, respectively. The C and N concentrations of plant samples were analyzed using an elemental analyzer (2400 II CHNS). The P concentration was analyzed using the molydate/ascorbic acid method after H₂SO₄-H₂O₂ digestion.
Important findings The C, N and P concentrations of different organs followed the order of: shoot (495.07 g·kg^-1) > twig (483.37 g·kg^-1) > fruit (480.35 g·kg^-1) > root (468.47 g·kg^-1) > leaf (466.33 g·kg^-1); leaf (22.27 g·kg^-1) > fruit (19.74 g·kg^-1) > twig (7.98 g·kg^-1) > shoot (4.54 g·kg^-1) > root (4.00 g·kg^-1) and fruit (2.85 g·kg^-1) > leaf (1.92 g·kg^-1) > twig (0.96 g·kg^-1) > root (0.52 g·kg^-1) > shoot (0.45 g·kg^-1), respectively. The ranges of the coefficient of variation (CV) for C, N and P concentrations were 1.71%-4.44%, 14.49%-25.50% and 11.46%-46.15%, respectively. Specifically, the C concentration was relatively high and stable, and the maximum CV values for N and P were found in roots. The N:P value of different organs varied from 7.12-12.41 and the minimum CV for N:P was found in twig, which indicated that N:P in twig had higher internal stability. In addition, correlation analysis indicated that the C concentration was significantly negatively correlated with N and P concentrations and correlation coefficients were -0.407 and -0.342, respectively. However, N concentration had dramatically positive correlation with P concentration and the correlation coefficient was 0.814. These results also could indicate that the C, N and P stoichiometric characteristics in the S. angustata shrub accorded with the homeostatic mechanism and growth rate hypothesis to some extent, the distributions of C, N and P concentrations were closely related to the function of the organs and it should be prudent to use ecological stoichiometric ratios to judge the condition of nutrient limitation at the species level.     

Oxidative DNA damage in tissues of English sole (Parophrys vetulus) exposed to nitrofurantoin

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2′deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r² = 0.59, P = 0.015) with the dose of NF (0.10 – 10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/μg DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/μg DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/μg DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.     

Benzoapyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice

Diesel exhaust particles (DEP) are a major source of air-borne pollution and are linked to increased risk of disease including lung cancer. Here we investigated effects of exposure to DEP on the frequency of DNA deletions, levels of oxidative DNA damage and DNA adduct formation during embryonic development in mice. Pregnant dams were orally exposed to various doses of DEP (500, 250, 125, 62.5, 31.25 mg/kg/day) at embryonic days 10.5–15.5. We determined the frequency of 70 kb DNA deletions spanning exons 6–18 at the p^un allele that results in black-pigmented spots in the unpigmented retinal pigment epithelium in the eyes of p^un/p^un offspring mice. DEP caused a significant increase in the frequency of DNA deletions. Levels of 8-OH deoxyguanosine indicating oxidative DNA damage were within the limits of the unexposed mouse embryos. ³³P post-labeling analysis revealed very low levels of DNA adducts in the embryo tissue. Thus, transplacental exposure to DEP resulted in a significant increase in the frequency of DNA deletions in the mouse fetus and such genetic alterations in the offspring may have pathological consequences later in life.     

Mutagenic effect of cadmium on tetranucleotide repeats in human cells

Cadmium is a human carcinogen that affects cell proliferation, apoptosis and DNA repair processes that are all important to carcinogenesis. We previously demonstrated that cadmium inhibits DNA mismatch repair (MMR) in yeast cells and in human cell-free extracts (H.W. Jin, A.B. Clark, R.J.C. Slebos, H. Al-Refai, J.A. Taylor, T.A. Kunkel, M.A. Resnick, D.A. Gordenin, Cadmium is a mutagen that acts by inhibiting mismatch repair, Nat. Genet. 34 (3) (2003) 326–329), but cadmium also inhibits DNA excision repair. For this study, we selected a panel of three hypermutable tetranucleotide markers (MycL1, D7S1482 and DXS981) and studied their suitability as readout for the mutagenic effects of cadmium.

We used a clonal derivative of the human fibrosarcoma cell line HT1080 to assess mutation levels in microsatellites after cadmium and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) exposure to study effects of cadmium in the presence or absence of base damage. Mutations were measured in clonally expanded cells obtained by limiting dilution after exposure to zero dose, 0.5 μM cadmium, 5 nM MNNG or a combination of 0.5 μM cadmium and 5 nM MNNG.

Exposure of HT1080-C1 to cadmium led to statistically significant increases in microsatellite mutations, either with or without concurrent exposure to MNNG. A majority of the observed mutant molecules involved 4-nucleotide shifts consistent with DNA slippage mutations that are normally repaired by MMR. These results provide evidence for the mutagenic effects of low, environmentally relevant levels of cadmium in intact human cells and suggest that inhibition of DNA repair is involved.     

我国北方几种玉米和设施菜地土壤的N2O和N2排放特征

为了探究旱地土壤施入氮肥后的气态氮(N₂O和N₂)损失规律,本研究通过室内好氧培养试验(60 d,25 ℃,80%孔隙含水量),运用¹⁵N同位素示踪技术,研究了4个玉米地土壤(哈尔滨、沈阳、栾城、寿光)和2个设施菜地土壤(沈阳、寿光)在施入尿素后的氮转化、N₂O和N₂排放动态。试验中尿素添加量为167 mg N·kg^-1,以模拟田间氮肥施用量200 kg N·hm^-2。结果表明: 在4个玉米地土壤中,尿素施用60 d内N₂O累积排放量为寿光(20 mg N·kg^-1)>栾城(14 mg N·kg^-1)>沈阳(5 mg N·kg^-1)>哈尔滨(0.5 mg N·kg^-1),N₂累积排放量为栾城(176 mg N·kg^-1)>沈阳(106 mg N·kg^-1)>寿光(75 mg N·kg^-1)>哈尔滨(12 mg N·kg^-1);在2个设施菜地土壤中,寿光土壤N₂O累积排放量(21 mg N·kg^-1)是沈阳(2 mg N·kg^-1)的10倍,而两个站点N₂累积排放量分别为28和24 mg N·kg^-1。不同土壤N₂O排放占两种气体排放总量的5%～40%,其中寿光土壤(30%～40%)显著高于其他样地土壤(1%～10%)。在土壤排放的N₂O和N₂中,土壤氮库分别贡献了56%和61%,高于添加当季氮肥的贡献率。相关分析表明,N₂O累积排放量与本底土壤pH呈正相关,说明土壤本底pH可能是调控不同旱地土壤N₂O和N₂排放的重要环境因子。在华北碱性土壤区,采用能降低土壤pH值的措施可能具有较好的气态氮减排效果。     

Induction of cytochrome P450 1A1 expression in captive river otters fed Prudhoe Bay crude oil: evaluation by immunohistochemistry and quantitative RT-PCR

Numerous studies have explored the relationships between exposure to a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons, and induction of cytochrome P450 1A (CYP1A) in different vertebrates. Few controlled studies, however, simulated chronic long-term exposure with repeated non-lethal sampling of the same individuals, which should better represent repeated exposure incidents in animals inhabiting polluted areas. In this study, we investigated the effects of chronic exposure to crude oil on levels of CYP1A1 in endothelial cells of skin biopsies and peripheral mononuclear blood cells in captive river otters (Lontracanadensis) using repeated sampling of the same individuals. We hypothesized that ingestion of oil would result in an increase in levels of CYP1A1 in both targets, and predicted that the relationship between prolonged exposure and expression of CYP1A1 would reach a plateau indicative of continuous detoxification of hydrocarbons. Fifteen wild-caught male otters were acclimated to captivity, and then fed diets containing no oil (control) or diets containing weathered crude oil at 5 mg day^-1 kg^-1 body weight (low-dose) and 50 mg day^-1 kg^-1 body weight (high-dose), at the Alaska Sealife Center in Seward, Alaska, USA. Expression of CYP1A1 was assessed with immunohistochemical analysis of CYP1A1 protein in skin biopsies and by quantitative RT-PCR analysis of CYP1A1 mRNA in mononuclear blood cells. Both assays revealed a decrease between capture and the transfer to captivity, indicating that the enclosure at the Alaska Sealife Center, and the food we offered to the otters were free of potential inducers of CYP1A1. During the exposure period, increases in CYP1A1 expression were registered by both techniques, followed by a decline in CYP1A1 after oil administration ended. Levels of endothelial CYP1A1 in the high-dose group were comparable to those recorded for wild river otters in PWS in 1996 and 1997. Levels of CPY1A1 mRNA in mononuclear blood cells, however, were well below levels recorded for river otters in Prince William Sound, and no correlation was detected between values obtained from the two methods. Thus, our results from this longitudinal study with repeated sampling of the same individuals provide support for the use of cytochrome P450 1A1 as a biomarker for hydrocarbon exposure. Nonetheless, our results also suggest that the induction process of CYP1A1 may be complicated and interacting with other processes in vivo. Such interactions may obscure our ability to describe specific, quantitative, predictable, dose-response relationships between exposure to hydrocarbons and induction of CYP1A1, which are required of reliable biomarkers. Evaluations of such interactions based on theoretical physiological models in live-animals merit further investigation.     

恩诺沙星和铜复合污染对蚯蚓消化酶活性的影响

从农田土壤畜禽粪源污染的实际出发,以典型兽药抗生素恩诺沙星(ENR)和饲料添加剂铜(Cu)为污染物,研究了两污染物单一/复合暴露对蚯蚓的毒性效应.结果表明: 单一污染暴露下,恩诺沙星(0.1～4 mg·kg^-1,28 d)对蚯蚓蛋白酶未产生显著影响,对纤维素酶和碱性磷酸酶产生了抑制作用,而对酸性磷酸酶产生了诱导效应;铜(20～200 mg·kg^-1,28 d)对蚯蚓的蛋白酶、纤维素酶和磷酸酶活性总体均表现为抑制效应.复合污染暴露下,两污染物对蚯蚓消化酶的影响以抑制效应为主,且对纤维素酶和酸性磷酸酶的抑制表现出毒性增加的协同效应.消化酶随暴露时间的响应动态规律为: 调整性反应(3 d)-激烈反应(7 d)-反应平复(14 d)-慢性暴露(28 d).慢性暴露结果显示,含高剂量(200 mg·kg^-1 Cu或4 mg·kg^-1 ENR)污染物的复合组更具生态风险性.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR, BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

The electrostatic interaction between the reaction-center bacteriochlorophyll derived from Rhodopseudomonas spheroides and mammalian cytochrome c and its effect on light-activated electron transport

1. By means of Q-switched ruby-laser flash excitation, the photooxidation of P870 in the reaction-center complex isolated from Rhodopseudomonas spheroides takes place within 1 μsec. The reduction of photooxidized P870 in the dark follows a first-order kinetics, with a pseudo first-order rate constant of 1.85×10⁸ l×mole^-1×sec^-1 and an activation energy of 6 kcal/mole.

2. Through an electrostatic interaction of the bacteriochlorophyll reaction-center complex and mammalian cytochrome c, an intimate contact between the two components resulted, and a collision-independent electron-transfer with a halftime of 25 μsec can be attained by laser-flash excitation. The absorbance changes at 870 and 550 nm indicated a good stoichiometry of the reaction. The oxidation of the c-type cytochrome in cells of Rps. spheroides (R-26 mutant) has a halftime of 12 μsec.

3. The portion of P870 which recovered rapidly was closely related to the mole ratio of cytochrome/P870. Complete recovery with a halftime of 25 μsec occurred when the cytochrome/P870 ratio was above approx. 10. At cytochrome/P870 ratios lower than 10, only the fraction of the reaction-center complex which have cytochromes bound at the active site can recover with the rapid decay time. Ultrafiltration measurements showed that each particle of the reaction-center complex can bind approx. 24 cytochrome molecules.

4. An electro static interaction is expected simply from the large difference between the isoelectric points of cytochrome c ( 10) and that of the reaction-center complex (4.1 measured by electro-focusing). The electro static interaction was further evidenced by the effects of pH, ionic strength, and by polylysine displacement of binding sites on the coupled oxidation of ferrocytochrome c by P870. From the limiting polylysine concentration giving complete blocking of cytochrome coupling, it was calculated that each reaction-center complex with a particle weight of 6.5×10⁵ contained approx. 500 negative charges.

5. Arrhenius plot of the first-order rate constants vs. the reciprocal absolute temperature yielded an activation energy of 12 kcal/mole for the cytochrome/P870 reaction, which is presumably the energy needed for cytochrome to achieve the most favorable orientation for the rapid electron transfer. Below the freezing temperature of the sample, the cytochrome reaction appeared to be uncoupled. The temperature dependence is consistent with the effect of viscosity on the reaction rate.

6. Double flash excitations spaced 200 μsec apart showed that at a cytochrome/P870 ratio of 24, the first flash caused maximum oxidation, indicating that all the reaction-center particles have at least one cytochrome attached to the active site. However, only 60% of the particles have a second cytochrome closely attached and capable of undergoing the rapid electron transport.     

稳定性铵态氮肥在黑土和褐土中的氮素转化特征

以稳定性氯化铵为氮源,采用室内培养的方法,研究0.20、0.50、1.00 g N·kg^-1干土3种浓度的稳定性铵态氮肥在黑土、褐土中的氮素转化特征.结果表明: 在褐土中,随着氯化铵添加量的增加,土壤中发生硝化作用的时间逐渐推迟,添加0.20、0.50 g N·kg^-1干土处理开始发生明显硝化反应的时间分别为第3、7天,在高浓度氮量(1.00 g N·kg^-1干土)添加下硝化作用受到明显抑制;在黑土中,各浓度氮量添加处理开始发生硝化反应的时间相同,均为第3天,且随着添加量的增加,硝化作用潜势逐渐减弱.只加铵态氮肥的处理中,添加0.20 g N·kg^-1干土的氯化铵氮肥在褐土和黑土中的硝化反应时间分别可维持3周和2周左右;添加0.50 g N·kg^-1干土的氯化铵氮肥在褐土和黑土中的硝化反应时间分别可维持4周和3周左右.与单施氯化铵相比,黑土和褐土在0.20、0.50 g N·kg^-1干土添加浓度下,按纯氮量的1.0%添加3,4-二甲基吡唑磷酸盐(DMPP)、4.0%添加二氰二胺(DCD)均能显著抑制硝化作用,降低硝态氮的含量,抑制硝化作用潜势.综上,在褐土中,随着氯化铵添加浓度增加,土壤硝化作用受到抑制效果大于黑土.在0.20、0.50 g N·kg^-1干土外源铵态氮时,添加抑制剂可以显著抑制铵态氮的硝化作用.因此室内硝化抑制剂培养试验时,建议铵态氮添加量不超过1.00 g N·kg^-1干土,以0.50 g N·kg^-1干土效果最好.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Mechanistic details for metathetical exchange between XCO (X = O and RN) and the tin(II) dimer, {Sn[N(SiMe3)2] (μ-OBu)}2

Metathetical exchange between carbon dioxide and the tin(II) dimer, {Sn[N(SiMe₃)₂](μ-OBu¹)}₂ (3) has been observed to cleanly produce the two new heteroleptic tin(II) dimers, Sn[N(SiMe₃)₂](μ-OBu^t)₂Sn(OSiMe₃) (6) and [Sn(OSiMe₃)](μ-OBu^t)]₂ (7]). In addition, reaction of 3 with I equiv, of tert-butylisocyanate (8), at 25°C, quantitatively provides 6, and with 2 equiv., quantitatively provides 7. Likewise 6 reacts with 1 equiv, of 8 to quantitatively provide 7. The mechanism for these latter processes has been investigated by low temperature ¹H NMR spectroscopy which reveals that metathetical exchange does not involve the tri-coordinate tin(II) centers of the dimeric structures, but rather, it occurs, in each case, via the transient monomeric tin(II) species, Sn[N(SiMe₃)₂](μ-OBu^t) (4), that undergoes metathesis to produce, initially the open dimer intermediate, Sn(OCNBu^t)(OSiMe₃)(μ-OBu^t)Sn(OBu^t) (OSiMe₃) (12), that is observed at −10°C. Subsequent redistribution reactions then generate the final products that are observed. Together, these mechanistic details provide additional support for the ‘monomeric tin(II)’ hypothesis proposed earlier for metathetical exchange between XCO and Sn[N (SiMe₃)₂]₂ (1).     

Biomarker responses in juvenile rainbow trout (Oncorhynchus mykiss) after single and combined exposure to low doses of cadmium, zinc, PCB77 and 17β-oestradiol

The objective of this study was to examine (i) the biochemical responses of rainbow trout exposed to sublethal water concentrations of the metals cadmium (Cd) (1.5 μg l^-1) and zinc (Zn) (150 μg l^-1); and (ii) the potential combined effects when applied in mixture (Cd/Zn) with and without co-exposure to model organic chemicals 3,3',4,4'-tetrachlorobiphenyl (PCB77) (1 mg kg^-1) and 17β-oestradiol (E2) (0.5 mg kg^-1). After 21 days of exposure, several biomarkers were assessed in the liver (enzymatic and non-enzymatic antioxidants, heat shock proteins [HSP70 and HSP60], ethoxyresorufin-O-deethylase [EROD]) and in the plasma (vitellogenin [Vtg], aminotransferases). Plasma aminotransferases were not affected, whereas the other biomarkers showed different patterns of response depending on the treatment. For example, Cd, and Zn to a lesser extent, induced an adaptive response in the liver shown by an increase in antioxidant defences (total glutathione [GSH], superoxide dismutase, Trolox equivalent antioxidant capacity [TEAC]), without any impairment of GSH redox status or induction of heat shock proteins. Antagonistic effects were observed in GSH-related biomarkers after Cd/Zn exposure. PCB77 strongly induced EROD activity, HSP70 and TEAC. Co-exposure with metals did not modulate significantly the effects of PCB77. E2 induced Vtg and inhibited liver antioxidants and basal EROD activity. These inhibitory effects were suppressed in fishes exposed to E2 + Cd/Zn, suggesting additive effects of E2 and metals. In addition, E2-induced Vtg was not altered by metals. Multivariate analyses confirmed some correlation between the biomarkers. The use of complementary biomarkers is necessary to discriminate different treatments and to highlight interactive effects.