Effects of curcumin on performance, antioxidation, intestinal barrier and mitochondrial function in ducks fed corn contaminated with ochratoxin A.

Curcumin has been attributed with antioxidant, anti-inflammatory, antibacterial activities, and has shown highly protective effects against enteropathogenic bacteria and mycotoxins. Ochratoxin A (OTA) is one of the major intestinal pathogenic mycotoxins. The possible effect of curcumin on the alleviation of enterotoxicity induced by OTA is unknown. The effects of dietary curcumin supplementation on OTA-induced oxidative stress, intestinal barrier and mitochondrial dysfunctions were examined in young ducks. A total of 540 mixed-sex 1-day-old White Pekin ducklings with initial BW (43.4±0.1 g) were randomly assigned into controls (fed only the basal diet), a group fed an OTA-contaminated diet (2 mg/kg feed), and a group fed the same OTA-contaminated feed plus 400 mg/kg of curcumin. Each treatment consisted of six replicates, each containing 30 ducklings and treatment lasted for 21 days. There was a significant decrease in average daily gain (ADG) and increased feed : gain caused by OTA (P<0.05); curcumin co-treatment prevented the decrease in BW and ADG compared with the OTA group (P<0.05). Histopathological and ultrastructural examination showed clear signs of enterotoxicity caused by OTA, but these changes were largely prevented by curcumin supplementation. Curcumin decreased the concentrations of interleukin-1ß, tumor necrosis factor-α and malondialdehyde, and increased the activity of glutathione peroxidase induced by OTA in the jejunal mucosa of ducks (P<0.05). Additionally, curcumin increased jejunal mucosa occludin and tight junction protein 1 mRNA and protein levels, and decreased those of ρ-associated protein kinase 1 (P<0.05). Notably, curcumin inhibited the increased expression of apoptosis-related genes, and downregulated mitochondrial transcription factors A, B1 and B2 caused by OTA without any effects on RNA polymerase mitochondrial (P<0.05). These results indicated that curcumin could protect ducks from OTA-induced impairment of intestinal barrier function and mitochondrial integrity.

Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-ß1).

OBJECTIVES: The role of mechanical stress and transforming growth factor beta 1 (TGF-ß1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known. METHODS: In this study, TGF-ß1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-ß1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours. RESULTS: A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-ß1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats. CONCLUSION: Mechanical stress-induced overexpression of TGF-ß1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-ß1 inhibitor can inhibit articular cartilage degradation.Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-ß1). Bone Joint Res 2018;7:587-594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1.

Effect of different dietary energy and protein sources on antioxidant status, fresh yolk fatty acid profile and microstructure of salted yolks in laying ducks.

The study investigated whether different dietary energy and protein sources affect laying performance, antioxidant status, fresh yolk fatty acid profile and quality of salted yolks in laying ducks. In all, 360 19-week-old Longyan ducks were randomly assigned to four diets in a factorial arrangement (2×2). The four diets consisted of two energy sources, corn (CO) or sorghum (SO) and two protein sources, soybean meal (SM) and rapeseed meal with corn distillers dried grains with solubles (RMD), and each treatment contained six replicates of 15 birds each. The experimental diets were isocaloric (metabolizable energy, 10.84 MJ/kg) and isonitrogenous (CP, 17%). The results showed that egg production, average egg weight, egg mass and feed conversion ratio were not affected by diets (P>0.05). Plasma contents of reduced glutathione (GSH), GSH/oxidized glutathione and total antioxidant capacity were lower (P<0.05) in ducks fed the RMD diets compared with those fed SM diets with a substantial increase (P=0.006) in plasma content of malondialdehyde (MDA). Egg yolks from ducks fed SO diets had higher proportions of polyunsaturated fatty acids (PUFA) and lower saturated and monounsaturated fatty acids compared with CO diets (P<0.001). Similarly, ducks fed RMD diets had a higher content of PUFA and n-6/n-3 ratio in fresh yolks (P<0.001), and increased salted yolk MDA, carbonylated proteins content and incidence of hard salted yolks (P<0.05) compared with SM diets. Scanning electron microscopy showed that salted yolks contained rougher polyhedral granules and fewer fat droplets, and were surrounded with a layer of bunchy fibers in ducks fed SO+RMD than those fed CO+SM diet. In conclusion, the current study showed that feeding laying ducks with diets containing SO or RMD reduced antioxidant capacity and increased egg yolk concentrations of PUFA. It appeared that egg yolks from ducks fed these diets were more sensitive to lipid peroxidation and protein oxidation during salting, and reduced the quality of salted yolks.

[Efficacy and safety of oseltamivir in children with suspected influenza: a multicenter randomized open-label trial].

Objective: To evaluate the efficacy and safety of oseltamivir in the treatment of suspected influenza in children. Method: A multicenter, randomized and open-label trial was conducted among 229 individuals with suspected influenza which were collected from the clinic of 5 hospitals in Guangdong province (Guangzhou Women and Children's Medical Center, Shenzhen Baoan District Maternity and Child Care Service Center, the Second Affiliated Hospital of Shantou University Medical College, Dongguan Maternity and Child Care Service Centre, Yuexiu District Children's Hospital of Guangzhou) from April to July 2015. They were randomized either to oseltamivir group (oseltamivir 30-75 mg, twice daily for 5 days) or control group who were given symptom relief medicines for 5 days. Result: No significant difference was found between two groups in influenza symptoms of the patients before the treatment(P>0.05). Altogether 229 individuals (114 in oseltamivir group, 115 in control group) were analyzed for efficacy, in which 73 individuals (42 oseltamivir, 31 control), 31.9%, were identified as influenza-infected through laboratory test. No significant difference was found between the two groups in the duration of fever although shortened. In the 229 individuals , the cumulative alleviation proportion between oseltamivir and control group was not significantly different (P>0.05): the median duration of illness was 69.9 hours (95% CI 65.3-91.5) in oseltamivir group and 75.4 hours (95%CI 63.9-91. 7) in control group; the median duration of fever was 40.4 hours (95%CI 31.5-53.4) in oseltamivir group and 44.0 hours (95%CI 33.2-50.0) in control group. In the 73 individuals, the cumulative alleviation proportion between oseltamivir and control group was significantly different (P<0.05). The median duration of illness was 61.2 hours (95%CI 48.0-121. 0) in oseltamivir group, being significantly shorter than that of 116.0 hours (95%CI 91.5-175.0) in control group. But it was not significantly different that the median duration of fever was 32.8 hours (95%CI 24.0-47.0 ) in oseltamivir group and 55.8 hours (95%CI 43.6-78.3 ) in control group (P>0.05). And the median duration of fever in 60 individuals (38 oseltamivir, 22 control) was significantly different between two groups(P<0.05), who had finished a course of taking oseltamivir in the 73 individuals, 34.8 hours (95%CI 24.0-48.5 ) in oseltamivir group being significantly shorter than that of 53.3 hours (95%CI 43.6-104.0 ) in control group. There was certain difference in side effects rate between the two groups (oseltamivir 10%, control 2%, P<0.05). The main side-effects were gastrointestinal symptoms (stomachache, diarrhea, poor appetite, vomiting). Conclusion: The duration of illness and fever in suspected influenza patients treated with oseltamivir was shorter than those in the patients treated with no oseltamivir, the difference was not statistically significant, when 31.9% was confirmed with positive result of virus test in suspected influenza in children. But in these patients with positive result of virus test, the duration of illness was significantly shortened with treatment with oseltamivir as compared with no treatment with oseltamivir, and it would be better if full oseltamivir course was completed for reducing the duration of fever. Oseltamivir treatment was safe with mild side effects.

Characterization of cold-induced remodelling reveals depot-specific differences across and within brown and white adipose tissues in mice.

AIM: Brown and beige adipose tissues dissipate energy in the form of heat via mitochondrial uncoupling protein 1, defending against hypothermia and potentially obesity. The latter has prompted renewed interest in understanding the processes involved in browning to realize the potential therapeutic benefits. To characterize the temporal profile of cold-induced changes and browning of brown and white adipose tissues in mice. METHODS: Male C57BL/6J mice were singly housed in conventional cages under cold exposure (4 °C) for 1, 2, 3, 4, 5 and 7 days. Food intake and body weight were measured daily. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous (sWAT) and epididymal white adipose tissue (eWAT) were harvested for histological, immunohistochemical, gene and protein expression analysis. RESULTS: Upon cold exposure, food intake increased, whilst body weight and adipocyte size were found to be transiently reduced. iBAT mass was found to be increased, whilst sWAT and eWAT were found to be transiently decreased. A combination of morphological, genetic (Ucp-1, Pgc-1α and Elov13) and biochemical (UCP-1, PPARγ and aP2) analyses demonstrated the depot-specific remodelling in response to cold exposure. CONCLUSION: Our results demonstrate the differential responses to cold-induced changes across discrete BAT and WAT depots and support the notion that the effects of short-term cold exposure are achieved by expansion, activation and increasing thermogenic capacity of iBAT, as well as browning of sWAT and, to a lesser extent, eWAT.

Evolution of viviparity in warm-climate lizards: an experimental test of the maternal manipulation hypothesis.

The maternal manipulation hypothesis for the evolution of reptilian viviparity has been claimed to apply to any situation where gravid females are able to maintain body temperatures different from those available in external nests, but empirical data that support this hypothesis are very limited. Here, we tested this hypothesis using gravid females of a warm-climate lizard, Mabuya multifasciata, by subjecting them to five thermal regimes for the whole gestation period. We found gravid females selected lower body temperatures and thermoregulated more precisely than did nongravid females. Offspring produced in different treatments differed in head size, limb length and sprint speed, but not in overall body size or mass. Variation in morphological traits of offspring was induced primarily by extreme temperatures. Sprint speed of offspring was more likely affected by the mean but not by the variance of gestation temperatures. Gravid females maintained more stable body temperatures than did nongravid females not because these temperatures resulted in the optimization of offspring phenotypes but because the range of temperatures optimal for embryonic development was relatively narrow. Our data conform to the main predictions from the maternal manipulation hypothesis that females should adjust thermoregulation during pregnancy to provide optimal thermal conditions for developing embryos and that phenotypic traits forged by maternal thermoregulation should enhance offspring fitness.

Dispersion modeling of PM10 released during decontamination activities.

The decontamination and decommissioning of radioactively contaminated structures and facilities, volume reduction of massive metal structures, and demolition of large concrete structures, result in release of large quantities of contaminants that become airborne and thus could be inhaled by workers and population living in the neighborhood. In order to provide adequate protection to the workers, adequate monitoring of the airborne particulates and proper models that predict the dispersion of the airborne contaminants are needed. The dispersion model will enable development of decision tools on the extent of decontamination that needs to be performed prior to dismantlement and the optimization of personal protective equipment requirements during D&D operations. The Gaussian plume dispersion model is used in this study to predict the dispersion of airborne particulate PM10 (dp<10 microm) released from: (1) a 35 m height contaminant plant where the plume is affected by the presence of 36 buildings around the emission source, (2) a building during decontamination and removal of process equipment and (3) demolition of contaminant building. The potential impact of PM10 on 180 receptors located at five downwind distances between 0.1 and 20 km around the emission source was performed. A short-term (1-48 h) prediction of average concentration of PM10 from point and area sources on receptors located at ground level was obtained. The concentrations of PM10 over 24h time period were compared to the U.S. air quality standards. The results obtained in the course of this study are used to predict the inhalation exposures of workers and population living in neighborhood.

Effects of bile acids on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced aberrant crypt foci and DNA adduct formation in the rat colon.

The effects of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci (ACF) in the rat colon were examined. The effect of these bile acids on DNA adduct formation by PhIP in the colon was then analyzed, since the main action of PhIP is the formation of DNA adducts and subsequent gene mutations. For the ACF study, male F344 rats were administered PhIP-HCl (75 mg/kg, 10 doses) by gavage, and a diet containing bile acid (0.4% DCA or UDCA) was provided from 3 days before the first dose of PhIP for 8 weeks. The mean number of ACF per colon of DCA, UDCA and controls were 9.9, 2.4 and 5.5, respectively. The ACF number was significantly increased by DCA and decreased by UDCA (P<0.001). To examine the effect of bile acids on DNA adduct formation, male F344 rats were fed a diet supplemented with bile acids (0.1 or 0.4% of DCA and UDCA) 7 days prior to the PhIP administration. All rats were administered a single dose of PhIP-HCl (50 mg/kg) by gavage and sacrificed 48 hours later. DNA adduct levels of the 0.1% UDCA, 0.1% DCA and controls were 2.93 (adducts/10(7) nucleotides), 2.65 and 1.10, respectively. Those of 0.4% UDCA, 0.4% DCA and controls were 1.64, 1.30 and 1.00, respectively. The PhIP-DNA adduct level was significantly increased by administration of 0.1% UDCA, 0.1% DCA (P<0.05) and 0.4% UDCA (P<0.01). The increasing effect of both DCA and UDCA on PhIP-induced DNA adduct formation was unexpected, and was not directly associated with ACF formation.

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

Effect of bile acids on formation of azoxymethane-induced aberrant crypt foci in colostomized F344 rat colon.

The effect of bile acids on the formation of azoxymethane induced aberrant crypt foci (ACF) was investigated using the fecal stream-excluded colons of colostomized F344 rats. The excluded colon was irrigated with saline or bile acids (1 mg/0.5 ml per day, 5 days/week) for 4 weeks. The mean numbers of ACF per colon in rats given cholic acid, deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), lithocholic acid, and ursodeoxycholic acid (UDCA) were 160.8, 118.2, 227.8, 150.7 and 87.3, respectively, while that of the control was 174.0. The number of ACF was significantly larger in CDCA, but smaller in UDCA and DCA-treated rats than the control (P<0.01). DCA did not induce apoptosis in the colon under the present conditions.

The p53-regulated cyclin-dependent kinase inhibitor, p21 (cip1, waf1, sdi1), is not required for global genomic and transcription-coupled nucleotide excision repair of UV-induced DNA photoproducts.

The p53 tumor suppressor gene is a transcriptional activator involved in cell cycle regulation, apoptosis, and DNA repair. We have shown that p53 is required for efficient nucleotide excision repair of UV-induced DNA photoproducts from global genomic DNA but has no effect on transcription-coupled repair. In order to evaluate whether p53 influences repair indirectly through cell cycle arrest following DNA damage or plays a direct role, we examined repair in vivo in human cells genetically altered to disrupt or regulate the function of p53 and p21. Both primary human fibroblasts and HCT116 colon carcinoma cells wild type for p53 but in which the p21 gene was inactivated through targeted homologous recombination showed no decrease in global repair of UV photoproducts. Human bladder carcinoma cells mutant for p53 and containing a tetracycline-regulated p21 cDNA showed no significant enhancement of repair upon induction of p21 expression. All of the cell lines, including the mismatch repair-deficient, MLH1 mutant HCT116 cells, were proficient for transcription-coupled repair. Clonogenic survival of HCT116 cells following UV irradiation showed no dependence on p21. Therefore, our results indicate that p53-dependent nucleotide excision repair does not require the function of the p21 gene product and is independent of p53-regulated cell cycle checkpoints.

DNA adduct formation by 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in rat colon.

A food-born carcinogen, 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) induces cancer in the rat colon. The mechanism for colonic DNA adduct formation leading to cancer by IQ was studied using a colostomized F344 rat model. In this model, the transverse colon of the rat was colostomized, which produced a fecal stream-positive proximal colon and a negative distal colon were produced. When IQ (50 mg/kg) was administered into the distal colon of the colostomized rats (n = 5), the ratio of the DNA adduct level of the distal colonic mucosa to the paired muscular layer 24 hr after dosage was 2.02, whereas that was 1.51 and 1.37 when IQ was administered into the stomach (n = 6) and the vein (n = 5), respectively. This suggested that luminal exposure of IQ induced DNA adduct formation. Since IQ (an amine form) has no reactivity toward DNA, these findings suggested that IQ was immediately activated in the absorbed mucosal cells and reacted with DNA. However, most of the IQ absorbed was metabolically activated in the liver, distributed by blood circulation, and formed DNA adducts in the colonic mucosa and muscular layer.

Identification of hepatic tamoxifen-DNA adducts in mice: alpha-(N(2)-deoxyguanosinyl)tamoxifen and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.

Tamoxifen-DNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifen-DNA adduct, four stereoisomers of alpha-(N:(2)-deoxyguanosinyl)tamoxifen N:-oxide 3'-monophosphate (dG(3'P)-N:(2)-TAM N:-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-alpha-acetoxytamoxifen N:-oxide in addition to four stereoisomers of alpha-(N:(2)-deoxyguano- sinyl)tamoxifen 3'-monophosphate (dG(3'P)-N:(2)-TAM) that was reported previously. Liquid chromatography-electrospray ionization-mass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M - H](-)), which corresponded to dG(3'P)-N:(2)-TAM N:-oxide. The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N:(2)-TAM N:-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/kg/day for 7 days by (32)P-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG(3'P)-N:(2)-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/10(8) nucleotides, which accounted for 53.3% of the total tamoxifen-DNA adducts. Lesser amounts of cis-dG(3'P)-N:(2)-TAM (2.8%) were also observed. An epimer of the trans-dG(3'P)-N:(2)-TAM N:-oxide (3.9 adducts/10(8) nucleotides) was detected as the third biggest adduct (7.2% of the total). The cis-dG(3'P)-N:(2)-TAM N:-oxide (0.4 adducts/10(8) nucleotides) accounted for 0.7% of the total. Thus, dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N:-oxide were identified in tamoxifen-treated mouse liver.

[Occurrence of Pagumogonimus veocularis in Fujian Province].

OBJECTIVE: To prove that Fujian Province is also a natural focus of Pagumogonimus veocularis(Pv). METHODS: The adult worms were obtained from a cat fed with Pv metacercariae. RESULTS: Pv were found in Jianou, Fujian Province. All 1,873 Semisulcospira libertina showed negative. The positive rate of Tricula fujianensis and Erhaia jianouenesis were 0.10% (1/695) and 0.25% (5/2,038), respectively. The main crab host was S. fujianensis. Ps alone and mixed infection with Pv were found in the Sinopotamon, the infection rates were 36.8% (43/117) and 20.5% (24/117), respectively. The numbers of the metacercariae were 806 and 40, respectively. A cat was infected with 12 metacercriae of Pv, eggs were found in the stool 56 days after infection, and 6 worms were found in the lungs 68 days after infection. CONCLUSION: Fujian is one of the natural focus of Pv, cat is the adequate host. The fluke was identified as Pv according to the characteristics of the metacercariae.

Tamoxifen-DNA adducts formed by alpha-acetoxytamoxifen N-oxide.

DNA adduct formation is assumed to be a major carcinogenic event, leading to the development of endometrial cancer in breast cancer patients taking tamoxifen and healthy women enrolled in a tamoxifen chemopreventive trial. To determine whether DNA adducts were formed by tamoxifen, trans- and cis-alpha-acetoxytamoxifen N-oxides were synthesized as model-activated forms via major tamoxifen metabolites, tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide. When alpha-acetoxytamoxifen N-oxide was reacted with human DNA, at least three DNA adducts were detected by (32)P-postlabeling coupled with HPLC. The total amount of DNA adducts formed by trans-alpha-hydroxytamoxifen N-oxide was 1.5-fold higher than that formed by the cis form. Both trans- and cis-alpha-acetoxytamoxifen N-oxide reacted with 2'-deoxyguanosine, resulting in the formation of three adducts (fr-1, fr-2-1, and fr-2-2). These products were studied using mass spectroscopy and proton magnetic resonance spectroscopy. fr-1 was identified as a mixture of the epimers of trans-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. fr-2-1 and fr-2-2 were determined to be epimers of cis-alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.

Mouse preproendothelin-1 gene. cDNA cloning, sequence analysis and determination of sites of expression during embryonic development.

Endothelin-1 (ET-1) is a peptide implicated in a wide variety of functions involving vascular and non-vascular systems. We have cloned the cDNA encoding the mouse prepro-endothelin-1 (PPET-1) and determined its nucleotide sequence. The putative PPET-1 peptide processing sites are all conserved and the deduced 21-amino-acid mature ET-1 peptide is identical to that of the rat, human, bovine, porcine and rabbit. Using the cloned cDNA as a probe for in situ hybridization, we detected PPET-1 mRNA in different tissues at different stages of mouse embryonic development. Embryos at a stage as early as 9.5 days postcoitum (E9.5) have very strong expression in the branchial epithelium, optic vesicle and the endothelial cells of large blood vessels, including the dorsal aorta and aortic arches. While the expression level in the branchial epithelium was decreasing towards the later stage of embryogenesis, the expression in the endothelial cells increased with age. At E10.5, PPET-1 mRNA was also detected in the otic vesicle as well as in the developing gut epithelium. At later stage of development, the expression of PPET-1 was primarily found in the vascular endothelial cells, cochlea, eye and the gut, with the highest level of PPET-1 mRNA in the endothelial cells of the lung. These data will be useful for analyzing the function of ET-1 in these organs.

Cloning, sequencing, and determination of the sites of expression of mouse sorbitol dehydrogenase cDNA.

Sorbitol dehydrogenase is one of the enzymes in the polyol pathway, which is thought to be implicated in the pathogenesis of diabetic complications. The cDNA encoding mouse sorbitol dehydrogenase was cloned from a liver library and its sequence was determined. The open reading frame encodes a product of 356 amino acids that shares high similarity with the human and rat liver sorbitol dehydrogenases (83% and 93% identity, respectively). The 3'-untranslated region contains a truncated L1Md repeat element inserted in reverse relative to the sorbitol dehydrogenase cDNA. Northern-blot hybridization showed that the testis has the highest level of expression, followed by kidney, liver, and lung. Low levels of expression were also observed in lens, brain, and skeletal muscle. In situ hybridization revealed that in the kidney, the highest concentration of sorbitol dehydrogenase mRNA is observed in the cortex, but is absent from the inner medulla. The parenchymal cells of the liver showed strong expression while the cells of the hepatic vasculature did not hybridize. The sorbitol dehydrogenase expression in the seminiferous tubules was mostly associated with the mature cells of the developing germ cells, confirming the usefulness of sorbitol dehydrogenase as an enzyme indicator for sexual maturation. The seminal vesicle, where most of the seminal fructose is produced, also showed a high level of expression in the epithelial cells. The mouse sorbitol dehydrogenase cDNA will be useful in the studies of the involvement of the polyol pathway in diabetic complications.

[The electrophysiologic effect of atenolol and nitrendipine on normal and ischemic rabbit myocardium in situ. II. Effects on effective refractory periods and its dispersion].

The study was carried out to determine the effective refractory period (ERP) in central part of ischemic region (CR), boundary of ischemic region (BR) and nonischemic region (NR), after occlusion of left ventricular branch of coronary artery in rabbit heart. The results turned out to be that atenolol produced significant prolongation of ERPs in all three regions before and after ligation of the coronary branch. Atenolol also caused significant reduction in dispersion of ERPs in ischemic heart. Nitrendipine, on the other hand, had no significant effects of all the parameters determined. We also found that slight changes in the absolute value of ERPs would result in much more greater changes in dispersion. So it could be inferred that the dispersion in ERPs among different regions of heart is of greater importance to the genesis and interruption of arrhythmias than ERPs themselves. Lastly, our results supported the view that there is a boundary region surrounding the central part of ischemic region, in which cells were moderately damaged and could be saved by proper treatment.

[The electrophysiologic effect of atenolol and nitrendipine on normal and ischemic rabbit myocardium in situ. I. Effects on transmembrane potential].

Transmembrane potential recording technique was achieved by using microelectrode with multiple impalement. The results showed that atenolol, both in normal and ischemic ventricular cells, produced significant lengthening of APD, but no apparent effects on APA, RS, OS and dv/dt max. Nitrendipine exerted no significant influences on all the parameters in normal and ischemic cells. Thus, it is suggested that beta-receptor blocker, atenolol possessed the characteristics of class III antiarrhythmic drug action on normal and ischemic rabbit heart in situ. This in situ action may provide some explanations for the mechanisms through which beta-blockers afford their beneficial effects of reducing the incidence of sudden death in postmyocardial infarction patients. Another interesting finding was that different parts of the repolarization phase of action potentials responded differently to interfering factors. According to the results obtained by analysing various parts of repolarization, we proposed three new parameters, APD0-50 (action potential duration from 0 to 50% of repolarization), APD50-100 (action potential duration from 50 to 100% of repolarization) and RI (repolarization index), to describe the different changes in various parts of repolarization. Discussion of the significance of these three is presented.

[The electrophysiologic effect of atenolol and nitrendipine on normal and ischemic rabbit myocardium in situ. III. Effects on ventricular fibrillation threshold].

This study was designed to assess myocardial vulnerability by measuring ventricular pacing threshold (VPT), multiple extrasystoles threshold (MET), ventricular tachycardia threshold (VTT) and ventricular fibrillation threshold (VFT) by means of the pulse train stimulating method. The results showed that: (1) VFT is a quantitative index of myocardial vulnerability: (2) MET, positively correlated with VFT, might become a substitute for VFT as an index of vulnerability to ventricular fibrillation: (3) atenolol, both in normal and ischemic myocardium, is able to make a significant elevation of VFT, which is considered as an antifibrillation effect: (4) nitrendipine exerted no apparent effects on all the parameters.