[Application evaluation of prefabricated rigid connecting bar in implants immediate impression preparation of edentulous jaw].

OBJECTIVE: To compare the operation complexity and accuracy of traditional splint impression technique and impression technique with prefabricated rigid connecting bar system for full-arch implants-supported fixed protheses in vitro. METHODS: Standard mandibular edentulous model with six implant analogs was prepared. The implants were placed at the bone level and multiunit abutments screwed into the implants. Two impression techniques were performed: the traditional splint impression technique was used in the control group, and the rigid connecting bar system was used in the test group. In the control group, impression copings were screwed into the multiunit abutments and connected with autopolymerizing acrylic resin. Open tray impression was fabricated with custom tray and polyether. In the test group, cylinders were screwed into the multiunit abutments. Prefabricated rigid bars with suitable length were selected and connected to the cylinders with small amount of autopolymerizing acrylic resin, and open tray impression was obtained. Impression procedures were repeated 6 times in each group. The working time of the two impression methods were recorded and compared. Analogs were screws into the impressions and gypsum casts were poured. The gypsum casts and the standard model were transferred to stereolithography (STL) files with model scanner. Comparative analysis of the STL files of the gypsum casts and the standard model was carried out and the root mean square (RMS) error value of the gypsum casts of the control and test groups compared with the standard model was recorded. The trueness of the two impression techniques was compared. RESULTS: The work time in the test group was significantly lower than that in the control group and the difference was statistically significant [(984.5±63.3) s vs. (1 478.3±156.2) s, P < 0.05]. Compared with the standard model, the RMS error value of the implant abutments in the test group was (16.9±5.5) µm. The RMS value in the control group was (20.2±8.0) µm. The difference between the two groups was not significant (P>0.05). CONCLUSION: The prefabricated rigid connecting bar can save the chair-side work time in implants immediate loading of edentulous jaw and simplify the impression process. The impression accuracy is not significantly different from the traditional impression technology. The impression technique with prefabricated rigid connecting bar system is worthy of clinical application.

Association of N-acetyltransferase-2 polymorphism with an increased risk of coronary heart disease in a Chinese population.

We investigated the possible correlations between N-acetyltransferase-2 (NAT2) gene polymorphisms and the risk of coronary heart disease (CHD). CHD patients (113) and healthy controls (118) were enrolled from the First People's Hospital of Yuhang between January 2013 and June 2014. The patients were divided into mild CHD (N = 72) and severe CHD (N = 41) subgroups. DNA samples were extracted and the distributions of NAT2 polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical characteristic indexes of severe CHD patients were also examined for relevant statistical analysis. WT, M1, M2, and M3 alleles were observed in both case and control groups. PCR-RFLP identified a wild-type homozygote, WT/WT; a mutant heterozygote, WT/Mx; and a mutant homozygote, Mx/Mx (x = 1, 2, and 3) variant of the NAT2 genotype. Mx/Mx differed significantly between case and control groups (P < 0.05); the frequencies of all four alleles did not differ significantly between case and control groups (P > 0.05). Slow acetylator genotype frequencies were notably higher in the case group than in the control group (P < 0.05). Individuals with the slow acetylator genotype were at 1.97-times higher risk of CHD and also displayed higher triglyceride and lower high-density lipoprotein cholesterol levels than those with the rapid acetylator genotype (P < 0.05). Therefore, the NAT2 polymorphism was believed to be associated with increased risk of CHD, with the NAT2 slow acetylator genotype serving as a risk factor for severe CHD in a Chinese population.

Effect of bradykinin on renal mesangial cell proliferation and extracellular matrix secretion.

Recent studies have found that bradykinin (BK) plays a role in delaying glomerulosclerosis, although the mechanism of this phenomenon remains unclear. Mesangial cell proliferation (MCP) and extracellular matrix (ECM) secretion are important mechanisms for glomerulosclerosis. This study investigated the impact of BK on the platelet-derived growth factor (PDGF)-induced proliferation of mesangial cells, and evaluated its correlations with the extracellular signal-related kinase (ERK) signaling pathway. The results showed that on its own, 10-1000 mg/L BK promoted MCP and ECM secretion and induced ERK phosphorylation. However, BK administration after PDGF pre-incubation inhibited PDGF-induced MCP, ECM secretion, and ERK phosphorylation. The BK B2 receptor-specific antagonist, HOE-140, and tyrosine phosphatase inhibitor (OV) effectively blocked the function of BK. In summary, these results demonstrated that BK has a bidirectional effect on MCP and ECM secretion: when used alone, it promoted effects on these phenomena, but these effects were inhibited when combined with PDGF. This suggests that the role of BK might be achieved through inhibiting activation of the PDGF-induced ERK1/2 pathway.

Effects of miR-150-5p on cerebral infarction rats by regulating the Wnt signaling pathway via p53.

OBJECTIVE: The aim of this study was to screen differentially expressed micro ribonucleic acids (miRNAs) in the plasma of patients with cerebral infarction (CI). In addition, the role of miR-150-5p in the incidence of CI is mainly explored via animal models and molecular biology experiments. PATIENTS AND METHODS: Blood samples were collected from hospitalized patients diagnosed with CI, including 15 CI patients and 15 non-CI patients as negative controls. Differentially expressed miRNAs in the plasma of these subjects were screened by microarray analysis. TargetScan was applied to predict the target genes of miR-150-5p, which were subjected to GO and pathway enrichment analyses using WebGestalt. Sprague-Dawley rats were randomly divided into Sham group (n=20), Control group (n=20), and Experimental group (n=20). CI model in rats was established in the latter two groups. Rats in Experimental group and Control group were intravenously injected with miR-150-5p mimics or miR-negative control (NC), respectively. The expressions of vital genes in the Wnt signaling pathway, including p53, Cyclin D1 (CCND1), c-Myc, ß-catenin (CTNNB1) and Survivin were detected by Western blot in rats at 3 d after injection. RESULTS: A total of 3,568 differentially expressed miRNAs were detected in the peripheral blood between CI patients and controls, whose 2,100 were upregulated, including miR-150-5p (p<0.05). The target genes of miR-150-5p were involved in molecular pathways, such as the Wnt signaling pathway, carcinogenesis, endocrine regulation, and infection. Compared with rats in Control group, the protein expression of p53 was downregulated (p<0.05), while CCND1, c-Myc, CTNNB1 and Survivin were upregulated (p<0.05) in Experimental group. CONCLUSIONS: MiR-150-5p regulates the Wnt signaling pathway and participates in cell proliferation and apoptosis by downregulating p53, which may be a potential mechanism of CI induction.

[Spatial clustering analysis and trend of liver cancer death rate in Shandong province, 1970-2013].

Objective: To explore the spatial clustering and trend of liver cancer mortality in different counties of Shandong province from 1970 to 2013, and provide scientific basis for the development of liver cancer prevention and control plan. Methods: Cancer mortality data were obtained from Shandong Death Registration System and three national death cause surveys in China. Mortality rate and age adjusted mortality rate were used to describe the trend of liver cancer in different years. Difference decomposing method was applied to estimate the contribution of demographic and non-demographic factors to the change of mortality. Software ArcGIS 10.2 was used for spatial analysis, and software SaTScan 9.4 was used for spatial clustering analysis on liver cancer mortality. Results: From 2011 to 2013, the crude mortality rate of liver cancer (29.89/100 000) in Shandong increased by 208.00% and 35.37% respectively compared with that during 1970-1974 (9.72/100 000) and 1990-1992 (22.08/100 000) and was similar to that during 2004-2005 (30.44/100 000). While age standardized mortality rate (ASMR) increased first and then decreased. The ASMR during 2011-2013 (12.62/100 000) increased by 60.97% compared with that during 1970-1974 and decreased by 22.38% and 21.81% compared with that during 1990-1992 and 2004-2005, respectively. According to the difference decomposition analysis on liver cancer mortality in different years, the contribution of population factors to the liver cancer mortality rate increased from 3.38% during 1990-1992 to 29.36% during 2004-2005 and 46.16% during 2011-2013. However, the contribution of non-population factors to the increase of liver cancer mortality decreased. According to the spatial distribution of liver cancer mortality, the crude mortality rate of liver cancer in different counties were quite different, ranging from 9.33/100 000 to 65.33/100 000. Using the spatial scanning statistical software to analyze the spatial clustering of liver cancer mortality, multi areas with high mortality rate of liver cancer were found, and they were mainly distributed in Jiaodong peninsula from 2011 to 2013, covering 20 counties (cities, districts) in Qingdao, Yantai and Weihai. The risk of liver cancer mortality in this area was 1.54 times higher than that in other areas. The spatial clustering distribution of liver cancer mortality during 1970-1974 was significantly different from that during 2011-2013, the areas with high mortality rate during 1970-1974 were mainly distributed in central and western Shandong. Conclusions: There were significant temporal and spatial distribution changes in the mortality rate of liver cancer in Shandong from 1970 to 2013. According to these trends and their geographical and spatial distribution, we should further explore the risk factors of liver cancer, and formulate feasible and area specific prevention and control measures for liver cancer.

MicroRNA-204-5p targets SOX11 to regulate the inflammatory response in spinal cord injury.

OBJECTIVE: The purpose of this study was to investigate whether microRNA-204-5p can regulate the inflammatory response of spinal cord injury (SCI) by targeting SOX11. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of microRNA-204-5p in patients with SCI. The mouse SCI model was established to detect the recovery of the grip strength of the upper and lower limbs. Then, the expression of microRNA-204-5p in these mice with SCI was detected by qRT-PCR, and the levels of the inflammatory factors Toll-like receptor 4 (TLR4) and iNOS were examined by Western blot. Subsequently, microRNA- 204-5p was overexpressed in the mouse SCI model using lentivirus, and the changes in mouse grip strength and the inflammatory factor levels were observed. SOX11 was then searched as the target gene of microRNA-204-5p through bioinformatics analysis, and its expression in patients or mice with SCI was examined using qRT-PCR. SOX11 expression was again detected after the overexpression or knockdown of microRNA-204-5p in cells. The binding of microRNA-204-5p to SOX11 was verified by dual-luciferase reporting assay. After microRNA-204-5p and SOX11 were co-overexpressed in cells, the levels of TLR4 and iNOS were analyzed. Furthermore, the changes in the grip strength were observed in mice with SCI after simultaneous up-regulation of microRNA-204-5p and SOX11. RESULTS: Micro-204-5p level was conspicuously decreased in the population with SCI. And the SCI mouse model showed that the upper and lower limb strength conspicuously decreased and began to recover after 7 days. During the seven days, microRNA-204-5p level in the SCI mice decreased with time, while the levels of the inflammatory cytokines TLR4 and iNOS conspicuously increased. After microRNA-204-5p was overexpressed in SCI mice, their upper and lower limb strength was conspicuously restored, while the levels of TLR4 and iNOS were also remarkably decreased. The bioinformatics analysis revealed that there exist some binding sites between microRNA-204-5p and SOX11, and we found that SOX11 expression was conspicuously enhanced in the plasma of the SCI patients. Meanwhile, the SOX11 level in SCI mice was also conspicuously increased, and it was time-dependent. The expression of SOX11 was decreased after the upregulation of microRNA-204-5p, while the opposite result was observed after the downregulation of microRNA-204-5p. In addition, the result of the dual-luciferase reporter gene assay revealed that microRNA-204-5p could bind to SOX11 in a targeted manner. Meanwhile, the up-regulation of SOX11 was partially relieved by the inhibitory effect of microRNA-204-5p on TLR4 and iNOS. Moreover, the simultaneous overexpression of SOX11 and microRNA-204-5p partially reversed the impact of the up-regulated microRNA-204-5p alone on the recovery of the upper and lower limb strength in SCI mice. CONCLUSIONS: The low expression of microRNA-204-5p in patients with SCI can affect the levels of the inflammatory cytokines TLR4 and iNOS and improve SCI by targeting SOX11.

MiR-325-3p promotes locomotor function recovery in rats with spinal cord injury via inhibiting the expression of neutrophil elastase.

OBJECTIVE: This study aims to investigate the potential function of miR-325-3p in vascular integrity and inflammatory response following spinal cord injury (SCI). MATERIALS AND METHODS: The protein levels of ANG-1, ANG-2, and caspase-3 in HUVECs incubated with 0, 100, 200, 400, and 800 ng/ml NE for 24 h were determined. The regulatory effect of overexpressed miR-325-3p on the protein levels of ANG-1 and ANG-2 was determined by Western blot. The SCI model in SD rats was established by spinal injury at T10. Subsequently, the relative levels of miR-325-3p, ANG-1, and ANG-2 were determined in SCI rats and controls. Furthermore, SCI rats were administrated with miR-325-3p mimics or negative control and the relative levels of miR-325-3p, ANG-1, and ANG-2 were examined as well. At day 14, the protein levels of iNOS and GFAP in SCI rats and those overexpressing miR-325-3p were detected. BBB (Basso, Beattie, and Bresnahan) locomotor rating scale was applied for evaluating the locomotor function recovery at day 1, 3, 7, 14, 21, and 28 following SCI. RESULTS: NE treatment in HUVECs downregulated ANG-1 and upregulated ANG-2 and caspase-3 in a dose-dependent manner. The overexpression of miR-325-3p upregulated NE-induced decreased the level of ANG-1 and downregulated NE-induced increased level of ANG-2. After the establishment of the SCI model in rats, the miR-325-3p level gradually decreased in SCI rats relative to controls in a time-dependent manner. ANG-1 level in SCI rats decreased to the lowest on the first day following SCI, and gradually increased at day 3, 5, and 7. ANG-2 level was firstly upregulated and achieved the peak on day 3, and then decreased at day 5 and 7. Moreover, SCI rats overexpressing miR-325-3p showed a higher level of ANG-1 and lower level of ANG-2 than those of SCI rats. Overexpression of miR-325-3p downregulated the protein levels of iNOS and GFAP in SCI rats. BBB scale showed elevated locomotor function recovery in SCI rats overexpressing miR-325-3p compared with SCI rats. CONCLUSIONS: MiR-325-3p protects the integrity of the vascular wall, reduces infiltration of inflammation, and improves locomotor function recovery at post-SCI.

[Trend of mortality and decomposition on malignant tumors in Shandong province, 1970-2013].

Objective: To describe the mortality trend of major malignant tumors in Shandong province, from 1970 to 2013. Methods: Data related to cancer mortality were obtained from the Shandong Death Registration System and three nationwide retrospective cause-of-death surveys. Trends of overall mortality and major causes of death were described using the indicators as: mortality rates and age-standardized mortality rates, through comparing the three large-scale mortality surveys in Shandong province. Difference decomposing method was applied to estimate the contribution of demographic and non-demographic factors for the change of mortality. Results: From 1970 to 2013, the crude mortality rate of malignant tumors in Shandong was increasing. The age standard mortality rate was increasing and then decreasing. The composition of cancer deaths in the all-cause-deaths was seen increasing and then decreasing as well. Both demographic and non-demographic factors contributed to the increase of crude cancer mortality rate. With the gradual increase of the proportion of population, its role exceeded the non-demographic factors. The age-standardized mortality rate of malignant tumors in 2011-2013 was lower than that in 2004-2005. Lung cancer mortality rose from the fifth to the first place, with an increase of 6.81 times from 1970-1974 to 2011-2013. Ranking of gastric cancer mortality dropped from first to the third place, with esophageal cancer dropped from second to the fourth. After adjusted by China's standard population in 1964, the mortality rate of lung cancer was still rapidly increasing, but the age-standardized mortality rates of esophageal cancer was gradually decreasing. The crude and age-standardized mortality rates of cervical cancer showed a rapid downward trend, reduced 87.00% and 93.00% respectively from 1970-1974 to 2011-2013. Conclusions: Malignant tumors were still major threats to the residents of Shandong province. The changing trend of different malignant tumors presented an inconsistent nature which called for different intervention strategies be carried out, accordingly.

[Acute toxicity and bio-distribution of silver nitrate and nano-silver with different particle diameters in rats].

Objective: To explore the acute toxic effect and the cumulative target organ of silver nitrate and nano-silver with two different particle diameters in rats. Methods: Thirty-six adult SD rats were divided into small particle size nano-silver group (SNS), large particle size nano-silver group (LNS), silver nitrate group (SN), and control group (C) according to the random number table, with 9 rats in each group. The rats of the four groups were respectively injected with 10 mg/mL nano-silver solution (particle diameter of 20 nm, prepared by saline) in silver dose of 30 mg/kg by tail vein for once, 10 mg/mL nano-silver solution (particle diameter of 100 nm, prepared by saline) in silver dose of 30 mg/kg, 1.67 mg/mL silver nitrate solution (prepared by glucose solution) in silver dose of 3 mg/kg, and 30 mg/mL polyvinylpyrrolidone solution (prepared by saline) in dose of 90 mg/kg. (1) Toxicity test. The general observation was performed within 14 days after injection, and the deviation between value of body mass before injection and each of that on post injection day (PID) 1, 7, and 14 were respectively recorded. On PID 1, 7, and 14, 3 rats of each group were harvested for determination of serum content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, and albumin by fully automatic biochemical analyzer. Then the rats were sacrificed immediately, and heart tissue, liver tissue, spleen tissue, lung tissue, kidney tissue, and brain tissue were collected to calculate the organ coefficient. Organ samples with obvious changes in organ coefficient were collected for histopathological observation by HE staining, with 3 samples in each group at each time point. (2) Bio-distribution. The specimens of heart, liver, spleen, lung, and kidney of rats from groups SNS, LNS, and SN were collected for detection of silver content by inductively coupled plasma mass spectrometry, with 3 samples in each group at each time point. Data were processed with analysis of variance of factorial design, LSD test, and Dunnett's T3 test. Results: (1) The general condition of rats in groups C and SN after injection were normal. The state of rats of groups SNS and LNS was poor with black secretion in the eye and other phenomena on PID 1, which recovered from PID 3 on. (2) The deviations between values of body mass before injection and that on PID 14 in rats of groups LNS and SN were significantly decreased as compared with deviation of group C (with P values below 0.01), but deviation of group SNS was not significantly changed (P>0.05). The deviations between values of body mass before injection and each of that on PID 1 and 7 in rats in the other three groups were similar to those in group C (with P values above 0.05). (3) Compared with those in group C, the serum content of total protein of rats in group SN on PID 1 was significantly decreased, and liver coefficient was significantly increased (with P values below 0.05). On PID 1, the serum content of ALT of rats in group LNS was (61.0±8.7) U/L, which was significantly higher than that in group C [(40.0±4.6) U/L, P<0.01]. Compared with those in group C [(126.0±3.5) U/L and 4.05±0.23], the serum content of AST of rats in groups SNS and LNS on PID 1[(249.7±107.2) and (237.0±38.3) U/L] was significantly increased, and liver coefficients (3.50±0.38 and 3.31±0.07) were significantly decreased, with P values below 0.05. Compared with those in group C [(69.2±4.8) U/L and 4.32±0.39], the serum content of AST of rats in groups SNS and LNS on PID 7 [(181.0±51.5) and (167.7±16.5) U/L] was also significantly increased, and liver coefficients (3.55±0.18 and 3.62±0.21) were also significantly decreased, P<0.05 or P<0.01. On PID 14, the four liver biochemical indexes in serum and all organ coefficients of rats in the other three groups were similar to those in group C (with P values above 0.05). (4) The liver of rats in group SN had slight degeneration on PID 1, the liver cells around the central vein of liver of rats in group SNS had slight degeneration on PID 7, and the liver cells got severely eosinophilic degeneration in liver of rats in group LNS on PID 7. There was no significant pathological change in the liver of rats in each group at the rest time points. (5) The silver content of lung and kidney in rats of group SNS on PID 1, that of spleen and kidney in rats of group LNS on PID 1, and that of heart and kidney in rats of groups LNS and SNS on PID 7 was significantly less than that of group SN (with P values below 0.05). The silver content of liver and spleen in rats of group SNS on PID 14 was significantly more than that of group SN (with P values below 0.05). Compared with that of group SN, the silver content of lung on PID 1 and liver on PID 7 in rats of group LNS was significantly increased (with P values below 0.05). On PID 14, there was no significant change in the silver content of all organs of rats between group SN and group LNS (with P values above 0.05). The silver content of heart, lung, and kidney on PID 1 and heart on PID 7 in rats of group LNS was significantly more than that of group SNS (with P values below 0.05). On PID 14, the silver content of each organ of rats in group SNS was close to that in group LNS (with P values above 0.05). Conclusions: Silver nitrate and nano-silver with two different particle diameters have a short acute toxic effect on the liver of rats, and the liver has certain ability of self-healing. Nano-silver is mainly accumulated in the liver. The distribution of nano-silver with large particle diameter in organs is more widely than that of nano-silver with small particle diameter.

Impact of carbapenem heteroresistance among clinical isolates of invasive Escherichia coli in Chongqing, southwestern China.

Although heteroresistance is common in a wide range of microorganisms, carbapenem heteroresistance among invasive Escherichia coli infections has not been reported. The objective of this study was to evaluate the clinical significance of carbapenem heteroresistance and to identify risk factors for its acquisition. A case-control study was conducted at a 3200-bed teaching hospital in Chongqing, southwestern China. Successive and non-duplicate nosocomial E. coli isolates (n = 332) were obtained from July 2011 to June 2013. Bloodstream isolates made up 50.6% of the strains collected. The rates of heteroresistance were 25.0% to imipenem, 17.2% to ertapenem, and 3.9% to meropenem. The population analysis profile revealed the presence of subpopulations with higher carbapenem resistance, showing MICs ranging from 2.0-128.0mg/L. Male gender, invasive intervention, antibiotic use and bacterial extended-spectrum ß-lactamase (ESBL) production contributed to invasive infections by carbapenem-heteroresistant E. coli (CHEC). The production of ESBL was identified as the common independent risk factor for heteroresistance to both ertapenem and imipenem. Pulsed-field gel electrophoresis revealed clonal diversity among the CHEC isolates. Most importantly, characterization of two successive E. coli strains isolated from the same patient indicated that carbapenem resistance evolved from heteroresistance. In conclusion, the high prevalence of heteroresistance to carbapenem among invasive E. coli merits great attention. Routine detection of ESBLs and the prudent use of imipenem and ertapenem are advocated. The early targeted intervention should be formulated to reduce CHEC infection and carbapenem resistance of E. coli.

Species differences in the metabolism and disposition of inhaled 1,3-butadiene and isoprene.

Species differences in sensitivity to carcinogenic effects from inhaled 1,3-butadiene might stem, at least in part, from differences in uptake, metabolism, and distribution of 1,3-butadiene. To examine this possibility, rats, mice, and monkeys were exposed to stepped concentrations of 14C-labeled 1,3-butadiene and the chemically related compound, isoprene. Respiratory data were collected during exposure and were used to determine fractional uptake. Rates and routes of excretion of retained radioactivity were also determined and blood levels of potentially toxic metabolites were measured. In some cases, the concentrations of hemoglobin adducts were determined. For rodents, the tissue distribution of metabolites was examined. Some results from these continuing studies to date are: a) mice achieve higher blood concentrations of reactive metabolites than do rats; b) blood levels of toxic metabolites are lower in monkeys than in rodents; c) uptake and retention of 1,3-butadiene is nonlinear in the range where long-term toxicity studies have been conducted; d) the efficiency of production of reactive metabolites decreases with increased inhaled concentrations of 1,3-butadiene; e) repeated exposure to 1,3-butadiene does not induce the metabolism of 1,3-butadiene in rodents; f) hemoglobin adducts of 1,3-butadiene are potential dosimeters of exposure; and g) rats inhaling isoprene produce reactive metabolites analogous to those produced during inhalation of 1,3-butadiene. The available data indicate that major differences in the biological fate of inhaled 1,3-butadiene occur among species, and these differences, at least in part, account for those in species sensitivity to the toxicity of inhaled 1,3-butadiene.

Contribution of intestinal microfloral metabolism to the total macromolecular covalent binding of 1-nitro-pyrene in the lung and liver of the rat.

1-Nitropyrene (NP) is a direct acting mutagen found in diesel exhaust and coal-combustion fly ash. The purpose of this study was to investigate the contribution of gut microfloral metabolism to the macromolecular covalent binding (MCB) of NP and/or its metabolites in lungs and liver of the rat. Normal and antibiotic treated rats were administered [14C]NP and MCB was quantitated at various times in lungs and livers. Abolition of gut microfloral metabolism by antibiotic treatment significantly altered total MCB in lungs. MCB in lungs of antibiotic-treated animals 4 h after oral administration of NP was 0.15 nmol NP equivalents/g and was significantly (P less than 0.05) decreased to less than one-half of control values (0.42 nmol NP equivalents/g). MCB in lungs of antibiotic-treated rats was no different from the controls 1 week after NP administration (0.1 nmol NP equivalents/g). Comparison of livers from control and antibiotic-treated rats demonstrated the same pattern of MCB as lungs but differences were not significant. These results reveal that metabolism by gut microflora may play a role in the activation and covalent binding of NP to macromolecules. However, the alteration of covalent binding observed after antibiotic treatment was a change in the time course of formation and breakdown of covalently bound forms and not an effect on the quantity of bound material remaining at 1 week indicating that gut microfloral metabolism is not an exclusive pathway for bioactivation of NP in the rat.

A new method for measuring covalent binding of chemicals to cellular macromolecules.

A new method has been developed for measuring the total covalent binding of metabolically activated compounds to cellular macromolecules. This method employs equilibrium dialysis, in the presence of the detergent sodium dodecyl sulfate (SDS), to remove unbound radiolabeled compound and its metabolites from cellular macromolecules. [14C] Bromobenzene (80 microM), [14C]aflatoxin B1 (5 microM) or 3-[14C]methylcholanthrene (100 microM) was incubated (37 degrees C) with primary hepatocytes or liver microsomes isolated from Fischer-344 rats. The covalent binding of 14C-radiolabel to hepatic or microsomal macromolecules was measured by SDS-equilibrium dialysis and compared with that measured by exhaustive extraction. After 1 h of incubation with hepatocytes or microsomes, 2--7 times more covalent binding was detected by SDS-equilibrium dialysis, than by exhaustive extraction. The radioactivity associated with these hepatic or microsomal macromolecules migrated to discrete positions on SDS-polyacrylamide disc gels. The non-dialysable radioactivity from incubations with [14C] bromobenzene could not be extracted with diethyl ether even after treatment of the dialysin with beta-glucuronidase-sulfatase or dilute acid. This was taken to indicate that the radioactivity in the dialysin did not include free bromobenzene or its metabolites, a conclusion supported by thin-layer chromatography analysis of the dialysin. The lower amount of covalent binding detected by exhaustive extraction may be related to the inability of trichloroacetic acid to quantitatively precipitate small molecular weight macromolecules. SDS-equilibrium dialysis is an easy, rapid and non-destructive technique for measuring covalent binding. The macromolecular integrity of the sample is maintained and allows further studies concerning the specificity of the covalent interactions.

The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity.

Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized. An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP), and 2-aminoanthracene (2AA). The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN). A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9). This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9. At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect-(MCA) acting mutagens. This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens. The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding. SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol. A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA. No detectable increase in SCE frequency was observed after repeated inhalation exposures. Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.

Disposition and metabolism of free and particle-associated nitropyrenes after inhalation.

The objective of this project was to determine the biological fate of 1-nitropyrene (NP) aerosols in rats. The results from these studies indicate that, over the range of aerosol concentrations tested, pathways for excretion of 14C-NP equivalents in urine and feces were independent of the exposure concentration of NP, either in its pure form or associated with diesel exhaust particles. In all cases, fecal excretion was the major route of elimination of 14C-NP equivalents, with about 2 times more excreted by this route than by urine. Fractional respiratory tract deposition of 14C-NP did not appear to be dependent on exposure concentration. In most cases, half-times for elimination of 14C in urine and feces were about 15 to 20 hours. In all exposures, 14C was widely distributed in the tissues examined. Analysis of the tissues for NP and metabolites indicated that within 1 hour after exposure greater than 90% of the 14C was associated with NP metabolites. Lungs of rats exposed to 14C-NP coated on diesel exhaust particles contained nearly 5 times more 14C than lungs from rats exposed to pure aerosols of 14C-NP (148 vs 29 pmole g lung) within 1 hour after exposure. This difference was increased to 80-fold at 94 hours after exposure (80 vs 1 pmole g lung). Long-term clearance half-times of 14C from various tissues were similar, with values of about 30 to 50 hours measured. Pre-exposure to diesel exhaust prior to exposure to NP may result in increased retention of a small fraction of the NP. Equilibrium organ concentrations predicted for tissues following continuous exposure to NP suggest that both low inhaled concentrations of NP and association of NP with insoluble diesel particles can result in an increased retention of NP in the lungs above what might be predicted using data obtained from animal studies using high concentrations of pure NP. The liver and kidneys are among the other organs predicted to contain the highest amounts of NP.

The role of ozone in tracheal cell transformation.

This project examined the potential role of ozone as a respiratory carcinogen by characterizing its ability to induce or modulate the preneoplastic transformation of rat tracheal epithelial cells. The chemical reactivity of ozone and the types of damage it can cause suggest that it may have a role in environmental carcinogenesis. Previous reports have described an increase in the incidence and number of lung tumors per animal in strain A mice exposed to ozone. However, the role of ozone in the development of the tumors has not been clear. Ozone also has been reported to act alone and synergistically with ionizing radiation to induce changes related to neoplasia in primary hamster embryo cells and in the mouse C3H/10T1/2 cell line in culture. Few other studies have examined the direct cytotoxic or transforming effects of ozone after in vivo or in vitro exposure of cells, and no studies have been reported on the comparative effects of ozone on respiratory cells exposed in vivo or in vitro. The induction of early preneoplastic changes in populations of rat tracheal epithelial cells by carcinogens can be detected and quantified in vitro after exposures in vivo or in vitro of tracheal epithelial cells. This cell culture and transformation system was used to characterize the transforming potency of ozone. Tracheal epithelial cells were isolated from Fischer-344/N rats that had been exposed for six hours per day, five days per week for one, two, or four weeks to 0, 0.12, 0.5, or 1.0 parts per million (ppm)* ozone (sea-level equivalents). Cell populations were examined in culture for increases in the frequency of preneoplastic variants. Rats exposed to ozone did not exhibit an increase in the frequency of preneoplastic tracheal cells, although exposed tracheas did exhibit dose-dependent morphological changes. Rat tracheal epithelial cells were given single, 40-minute in vitro exposures to concentrations of ozone that did not result in any detectable decrease in colony-forming efficiency (approximately 0.7 ppm) and to concentrations that resulted in approximately a 40% decrease (approximately 10 ppm). Exposed cultures were examined for increases in the frequency of preneoplastic variants. The results of these experiments, like those for the in vivo experiments described above, suggest that a single ozone exposure does not induce preneoplastic variants of rat tracheal epithelial cells. In contrast, cultures of rat tracheal cells exposed to 0.7 ppm ozone twice weekly for about five weeks exhibited approximately a twofold increase in the frequency of preneoplastic variants compared with control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Mucosal swabs detect HPV in laryngeal papillomatosis patients but not family members.

Seven patients, aged 2-7 years, with active recurrent respiratory papillomatosis (RRP) attending the University of Michigan Pediatric Otolaryngology Clinic were studied to determine if human papillomavirus (HPV) is harbored in sites of the upper aerodigestive tract other than in the laryngeal papilloma itself. We also determined if close family members had detectable virus in their oral cavities. Noninvasive swabs of buccal mucosa, posterior pharynx, nasal vestibule, and tonsillar pillar of patients, as well as buccal mucosa and posterior pharyngeal swabs of family members were studied. Swabs of the patients' papillomas served as the positive controls. HPV was detected using polymerase chain reaction (PCR) amplification and Southern hybridization techniques. Six of seven patients had detectable HPV in papilloma and endolaryngeal swabs. Four were HPV type 6, and two were HPV type 11. The patient whose swab was negative for HPV was found to be biopsy negative for papilloma 3 weeks after a single laser excision which was performed 6 months prior to the endolaryngeal swab. HPV types 16, 18 and 31 were not found in any of the patients. No swabs from other sites in patients or family members were HPV positive despite the presence of adequate DNA in the swabbed material for successful amplification of beta-actin sequences. The absence of HPV (other than in the papilloma itself) in the upper aerodigestive tract of patients and caregivers is consistent with the absence of reported cases of horizontal transmission to siblings or other family members. The findings are also consistent with the conventional view that juvenile respiratory HPV is transmitted vertically from vaginal condylomas in the mother.

Evaluation of prenatal nutrition counseling: maternal nutrition status and infant birthweight.

A prospective study on the effect of prenatal nutrition counseling on maternal nutrition status and infant birthweight was conducted at an antenatal care clinic by comparing a group of 80 women who attended nutrition counseling sessions with another group of 63 women who did not participate in nutrition counseling (controls). The daily intake of protein, calcium, iron, retinol, and riboflavin in the counseled group was higher than that in the control group. Moreover, the daily intake of nutrients of the counseled women met the recommended dietary allowance. Blood constituent determinations revealed that the levels of serum total protein, albumin, vitamin A, vitamin E, zinc, copper, magnesium, and hemoglobin in the blood of mothers and in umbilical blood at delivery were higher in the counseled group than in the control group (P less than 0.01). The women receiving counseling had fewer low-birthweight infants (1.52% vs 2.70%) and the incidence of maternal anemia was 39.1% against 55.6%, a significant difference (P less than 0.01).