[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.

[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.

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.

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.

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.

[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.

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.

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.

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.

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.

Thirteen-week, repeated inhalation exposure of F344/N rats and B6C3F1 mice to ferrocene.

Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile compound used as a chemical intermediate, a catalyst, and an antiknock additive in gasoline. This organometallic chemical is of particular interest because of its structural similarities to other metallocenes, some of which are carcinogenic. F344/N rats and B6C3F1 mice were exposed to 0, 3.0, 10, and 30 mg ferrocene vapor/m3, 6 hr/day, 5 days/week, for 13 weeks. During these exposures, no rats or mice died, nor were any clinical signs of ferrocene-related toxicity observed. At the end of the exposures, male rats exposed to the lowest and highest level of ferrocene had decreased body weight gains compared to filtered-air-exposed control male rats, while body weight gains for all groups of both ferrocene- and filtered-air-exposed female rats were similar. Male mice exposed to ferrocene had no differences in body weight gains, compared to controls, but female mice had decreases in body weight gains at the 10 and 30 mg/m3 exposure levels. There were exposure concentration- and exposure-time-related increases in lung burdens of iron. The mean iron lung burden in rats exposed to 30 mg ferrocene vapor/m3 for 90 days was four times greater than the burden in control rats. No exposure-related changes in respiratory function, lung biochemistry, bronchoalveolar lavage cytology, total lung collagen, clinical chemistry, and hematology parameters were observed. This suggests that the accumulations of iron in lung did not cause an inflammatory response nor any functional impairment of the lung. There were no indications of developing pulmonary fibrosis nor of any hematologic toxicity. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Exposure-related histopathologic alterations, primarily pigment accumulations, were observed in the nose, larynx, trachea, lung, and liver of both species, and in the kidneys of mice. Lesions were most severe in the nasal olfactory epithelium where pigment accumulation, necrotizing inflammation, metaplasia, and epithelial regeneration occurred. Nasal lesions were observed in all ferrocene-exposed animals and differed only in severity, which was dependent on the exposure concentration. Histochemical stains of these target tissues showed the presence of iron ions. The results suggest that the mechanism of ferrocene toxicity may be the intracellular release of ferrous ion through ferrocene metabolism, followed by either iron-catalyzed lipid peroxidation of cellular membranes or the iron-catalyzed Fenton reaction to form hydroxyl radicals that directly react with other key cellular components, such as protein or DNA.

Functional organization of the Bel-1 trans activator of human foamy virus.

Human foamy virus encodes a 300-amino-acid nuclear regulatory protein termed Bel-1 that is required for human foamy virus replication in culture. Bel-1 is a potent trans-activator of gene expression directed by the homologous HFV long terminal repeat as well as the long terminal repeat of human immunodeficiency virus type 1. We have used mutational analysis to define several discrete functional domains within Bel-1. The C-terminal approximately 50 amino acids of Bel-1 are shown to be essential for Bel-1 activity but can be effectively substituted by the C-terminal activation domain of VP16. We therefore conclude that the Bel-1 C terminus forms part of an activation domain. Mutations within a central, approximately 100-amino-acid segment of Bel-1 preclude trans-activation by either Bel-1 or the Bel-1/VP16 chimera. These sequences are therefore proposed to direct the interaction of Bel-1 with its viral DNA target sequences. A short Bel-1 segment located between the activation and binding domains is shown to mediate the nuclear localization of this regulatory protein. Although the functional organization of Bel-1 therefore appears comparable to that reported for other eukaryotic transcriptional activators, Bel-1 does not contain sequences homologous to known transcriptional activation or DNA-binding motifs.

Biological markers of exposure to benzene: S-phenylcysteine in albumin.

Results of experiments in our laboratory have shown that benzene is metabolized by animals in part to an intermediate that binds to cysteine groups in hemoglobin to form the adduct S-phenylcysteine (SPC). These results suggested that SPC in hemoglobin may be an effective biological marker for exposure to benzene. However, we could not detect SPC in the globin of humans occupationally exposed to benzene concentrations as high as 28 p.p.m. for 8 h/day, 5 days/week. As another approach, we examined the binding of benzene to cysteine groups of a different blood protein, albumin. To facilitate the process, a new method for the precipitative isolation of albumin from plasma was also developed. The isolated albumin was analyzed for SPC by isotope dilution GC-MS. We used this approach to measure SPC in the albumin of F344/N rats exposed by gavage to 0-10,000 mumol/kg benzene. Amounts of albumin-associated SPC increased as a function of dose, followed by a leveling off in the amount of SPC seen at doses greater than 1000 mumol/kg. Levels of SPC were measured in humans occupationally exposed to average concentrations of 0, 4.4, 8.4 and 23 p.p.m. benzene 8 h/day, 5 days/week. Of nine controls, seven had levels of SPC below the limit of detection (0.1 pmol SPC/mg albumin). SPC increased in the exposed groups linearly, giving a statistically significant slope (P less than 0.001) of 0.044 +/- 0.008 pmol/mg albumin/p.p.m. with an intercept of 0.135 +/- 0.095 pmol/mg albumin. From this study, we conclude that SPC in albumin may prove useful as a biomarker for benzene exposure.

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)

S-phenylcysteine formation in hemoglobin as a biological exposure index to benzene.

Benzene is metabolized to intermediates that bind to hemoglobin, forming adducts. These hemoglobin adducts may be usable as biomarkers of exposure. In this paper, we describe the development of a gas chromatography/mass spectroscopy assay for quantitating the binding of the benzene metabolite, benzene oxide, to cysteine groups in hemoglobin. We used this assay to study the hemoglobin adduct, S-phenylcysteine (SPC), in the blood of rats and mice exposed to benzene either by inhalation or by gavage. We were able to detect SPC in the hemoglobin of exposed rats and mice, to show the linearity of the exposure dose-response relationship, and to establish the sensitivity limits of this assay. For the same exposure regime, rats showed considerably higher levels of SPC than did mice. As yet, we have not been able to detect SPC in the globin of humans occupationally exposed to benzene. We attempted to determine whether the SPC found in hemoglobin originated from the metabolism of benzene within or outside of the red blood cell. We hypothesized that the greatest red blood cell metabolism would be associated with peripheral reticulocytes, which retain high metabolic capacity. After exposing rats to benzene, we isolated the red blood cells and used discontinuous Percoll gradients to fractionate them into age groups. No differences in SPC levels were found among any of the fractions, suggesting that the SPC found in globin originates from the metabolism of benzene to benzene oxide in a location external to the red blood cell. To our knowledge, this is the first demonstration of the nonenzymatic binding of the benzene metabolite, benzene oxide, to protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicokinetics of inhaled 1,3-butadiene in monkeys: comparison to toxicokinetics in rats and mice.

1,3-Butadiene is a potent carcinogen in mice and a weaker carcinogen in rats. People are exposed to butadiene through its industrial use--largely in rubber production (over 3 billion pounds of butadiene were produced in 1989)--and because it is common in the environment, occurring in cigarette smoke, gasoline vapor and in the effluents from fossil fuel incineration. Epidemiological studies have provided some evidence for butadiene carcinogenicity in people. Differences in the uptake and metabolism of inhaled butadiene between rodents and primates, including people, might be reflected in differences in its toxicity. In order to compare uptake and metabolism in primates to that in rodents--for which data were already available--we exposed cynomolgus monkeys (Macaca fascicularis) to 14C-labeled butadiene at concentrations of 10.1, 310 or 7760 ppm for 2 hr. Exhaled air and excreta were collected during exposure and for 96 hr after exposure. The uptake of butadiene as a result of metabolism was much lower in monkeys than in rodents. For equivalent inhalation exposures, the concentrations of total butadiene metabolites in the blood were 5-50 times lower in monkey than in the mouse, the more sensitive rodent species, and 4-14 times lower than in the rat. If the toxicokinetics of butadiene in people is more like that of the monkey than that of rodents, then our data suggest that people will receive lower doses of butadiene and its metabolites than rodents following equivalent inhalation exposures to butadiene. This has important implications for assessing the risk to humans of butadiene exposure based on animal studies.

Two-week, repeated inhalation exposure of F344/N rats and B6C3F1 mice to ferrocene.

Ferrocene (dicyclopentadienyl iron; CAS No. 102-54-5) is a relatively volatile, organometallic compound used as a chemical intermediate, a catalyst, and as an antiknock additive in gasoline. It is of particular interest because of its structural similarities to other metallocenes that have been shown to be carcinogenic. F344/N rats and B6C3F1 mice were exposed to 0, 2.5, 5.0, 10, 20, and 40 mg ferrocene vapor/m3, 6 hr/day for 2 weeks. During these exposures, there were no mortality and no observable clinical signs of ferrocene-related toxicity in any of the animals. At the end of the exposures, male rats exposed to the highest level of ferrocene had decreased body-weight gains relative to the weight gained by filtered air-exposed control rats, while body-weight gains for all groups of both ferrocene- and filtered air-exposed female rats were similar. Male mice exposed to the highest level of ferrocene also had decreased body-weight gains, relative to controls, while female mice had relative decreases in body-weight gains at the three highest exposure levels. Male rats had a slight decrease in relative liver weight at the highest level of exposure, whereas no relative differences in organ weights were seen in female rats. Male mice had exposure-relative decreases in liver and spleen weights, and an increase in thymus weights, relative to controls. For female mice, relative decreases in organ weights were seen for brain, liver, and spleen. No exposure-related gross lesions were seen in any of the rats or mice at necropsy. Histopathological examination was done only on the nasal turbinates, lungs, liver, and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Globin adducts of benzo[a]pyrene: markers of inhalation exposure as measured in F344/N rats.

We have explored methods for determining benzo[a]pyrene (BaP) dosimetry by measuring adduction levels to F344/N rat blood hemoglobin, and have refined and validated an assay that measures the in vivo binding of the 7,8-diol 9,10-epoxide metabolite of BaP to globin. The assay for BaP-globin adducts was based on the release of tetrahydroxy-BaP (BaP-tetrols) from globin by mild acid hydrolysis. After extensive isolation, BaP-tetrols were quantitated by high-pressure liquid chromatography using fluorescence detection. BaP-tetrol levels were measured in rats dosed intraperitoneally with 242, 71 and 24 mumol BaP kg-1 body weight in corn oil. The formation of BaP-tetrols was not linear with dose. The lowest dose yielded adduct levels that represented the limits of sensitivity for the method, as performed in this laboratory. Once this limit of sensitivity was established, the potential use of the assay was assessed by measuring the radiochemical binding of inhaled [14C]BaP or its metabolites to the globin of F344/N rats. Rats were exposed for 4 h per day, 1 day per week, for 12 weeks to pure aerosols of [14C]BaP at a level of 2 mg m-3. At the conclusion of exposure, rats were sacrificed and globin was isolated. The extent of [14C]BaP binding to the globin was determined by liquid scintillation spectrometry. Rats exposed to aerosols of [14C]BaP had statistically increased levels of binding to globin, and the levels were comparable to those observed previously after intragastric administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of inhaled isoprene in B6C3F1 mice.

Isoprene (2-methyl-1,3-butadiene) is the monomeric unit of widely occurring natural products called terpenes. Isoprene is widely used in industry with nearly 1.1 million pounds produced in the United States in 1987. The purpose of this investigation was to determine the toxicokinetics of inhaled isoprene in B6C3F1 mice and to compare the data to previously published toxicokinetic data in F344 rats (A. R. Dahl, L. S. Birnbaum, J. A. Bond, P. G. Gervasi, and R. F. Henderson, 1987. Toxicol. Appl. Pharmacol. 89, 237-248). The comparative toxicokinetics in the two species will be useful for extrapolation of rodent toxicity data to humans. Male B6C3F1 mice were exposed to nominal concentrations of 20, 200, and 2000 ppm isoprene or [14C]isoprene for up to 6 hr. For all exposures, steady-state levels of isoprene were reached rapidly (i.e., within 15 to 30 min) after the onset of exposure. The mean (+/- SE) steady-state blood levels of isoprene (identified by headspace analysis) for the 20, 200, and 2000 ppm exposures were 24.8 +/- 3.3, 830 +/- 51, and 6800 +/- 400 ng isoprene/ml blood, respectively. At the two higher exposure concentrations, the increases in blood levels of isoprene were proportional to the increases in air concentrations of isoprene. There was approximately a 2.3-fold decrease in the retained 14C/inhaled 14C ratio with increasing exposure concentration. Depending on the exposure concentration, from 52% (20 ppm isoprene) to 73% (2000 ppm isoprene) of the metabolite-associated (nonisoprene) radioactivity was excreted in the urine over a 64-hr postexposure period. 14CO2 exhalation after the end of the 6-hr exposure was minimal (2%) at the 20 ppm exposure and increased up to 18% at the higher isoprene exposure concentrations. These data suggest that metabolism of isoprene in mice is nonlinear within the range of exposure concentrations used in this study. Hemoglobin adduct formation reached near-maximum between 200 and 2000 ppm isoprene exposure concentration, consistent with our conclusion that pathways for metabolism of isoprene were saturated. Isoprene metabolites were present in blood after inhalation of isoprene at all concentrations studied. There were substantial differences in the toxicokinetics of inhaled isoprene in mice compared to rats. In mice, fractional retention of inhaled isoprene, which reflects, in part, metabolism of isoprene, was linearly related to exposure concentrations up to 200 ppm but decreased at 2000 ppm; in rats, fractional retention of inhaled isoprene decreased with increasing exposure concentration over a range of exposures from 8 to 1500 ppm.(ABSTRACT TRUNCATED AT 400 WORDS)