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Atherosclerosis is a multifactorial disease that is influenced by both genetic and environmental factors. Recent epidemiological studies have shown that the combination of elevated VLDL/LDL concentrations and elevated fibrinogen levels results in a strong increase of the risk for cardiovascular disease as compared to the individual risk factors. In humans, controlled studies on the relative contribution of the different factors are hampered by the heterogeneity in both environmental and genetic factors. To circumvent these limitations the APOE3-Leiden transgenic mice model was developed. This model provides the opportunity to induce, modulate and measure atherosclerosis in a quantitative and standardized manner. The causal relation between increased VLDL/LDL levels and atherosclerosis has been well established, whereas for fibrinogen such a causative relationship is still uncertain. Because fibrinogen is an acute-phase protein, we studied the possibility that plasma fibrinogen is a marker of the disease and becomes elevated as a consequence of inflammatory reactions that occur during the development of atherosclerotic plaques. The APOE3-Leiden mice were put on high cholesterol diet and at time intervals ranging from 4 to 30 weeks the plasma fibrinogen concentration was measured by a clotting rate assay and an ELISA. The progression of atherosclerosis in these mice was analyzed by histochemical methods. The fibrinogen levels in the plasma of APOE3-Leiden mice on a high cholesterol diet did not change with time. The atherosclerosis measured in the APOE3-Leiden mice on the high cholesterol diet ranged from no atherosclerosis to the presence of foam cells and the development of fatty streaks. The plasma fibrinogen concentration in APOE deficient mice after 15 weeks on a high cholesterol diet was the same as in wild-type mice. The atherosclerosis in the APOE deficient mice ranged from intermediate lesions to severe plaque formation whereas the wild-type mice showed no signs of atherosclerosis. These results indicate that the plasma fibrinogen concentration in APOE3-Leiden mice and in APOE deficient mice is not elevated secondarily to an early, intermediate, or advanced stage of atherosclerotic plaque formation. Copyright © 1997 Published by Elsevier Ltd.

Elevated plasma cholesterol, a well-known risk factor for cardiovascular diseases, is the result of the activity of many genes and their encoded proteins in a complex physiological network. We aim to develop a minimal kinetic computational model for predicting plasma cholesterol levels. To define the scope of this model, it is essential to discriminate between important and less important processes influencing plasma cholesterol levels. To this end, we performed a systematic review of mouse knockout strains and used the resulting dataset, named KOMDIP, for the identification of key genes that determine plasma cholesterol levels. Based on the described phenotype of mouse knockout models, 36 of the 120 evaluated genes were marked as key genes that have a pronounced effect on the plasma cholesterol concentration. The key genes include well-known genes, e.g., Apoe and Ldlr, as well as genes hardly linked to cholesterol metabolism so far, e.g., Plagl2 and Slc37a4. Based on the catalytic function of the genes, a minimal conceptual model was defined. A comparison with nine conceptual models from literature revealed that each of the individual published models is less complete than our model. Concluding, we have developed a conceptual model that can be used to develop a physiologically based kinetic model to quantitatively predict plasma cholesterol levels. © 2010 Elsevier B.V. All rights reserved.

The recent introduction of the phenyl-β-D-galactopyranoside (P-gal)-based positive-selection system for screening of λlacZ phages originating from the λlacZ transgenic mouse (Muta Mouse) has made the determination of mutant frequencies (MF) a much simpler task. Previously, MF data from these mice have been collected by means of the 5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside (X-gal) colour-screening procedure. To determine whether data obtained with the two systems are comparable, the MF in h phages recovered from liver and brain of transgenic mice treated with N-ethyl-N-nitrosourea (ENU) and liver of benzo(a)pyrene (B(α)P)-treated mice was determined with both procedures. For the livers of mice treated with ENU, both methods yielded approximately the same MF values. No induction of mutants, relative to the control animals, was seen after 1.5 h, but a clear 4-fold increase was measured with both assays at the 14-day time point. No induction of mutants was found in the brain with either method. In the B(α)P-treated mice, both methods showed a substantial induction in MF after 21, 28 and 35 days. The values generated by the X-gal and P-gal methods were not significantly different, with the exception of the 35-day post-treatment point that appeared higher in the X-gal assay. When the mutants isolated by use of the X-gal method were tested in the P-gal assay, a number of these did not turn up as mutants, and the significance disappeared, In conclusion, the data obtained with the two screening procedures agree to such an extent as to permit a direct comparison between the earlier results generated with X-gal and P-gal values generated with the new positive-selection method. This is likely to apply also to other organs and mutagens than those studied here.

Chemicals/CAS: misonidazole, 13551-87-6; Nitroimidazoles; Radiation-Sensitizing Agents

Pectin-derived acidic oligosaccharides (pAOS) are non-digestible carbohydrates to be used in infant formulae and medical nutrition. To support its safety, the genotoxic potential of pAOS was evaluated. pAOS was not mutagenic in the Ames test. Positive results were obtained in the chromosome aberration test only at highly cytotoxic concentrations. The effects obtained in the mouse lymphoma test were equivocal; pAOS was not mutagenic in vivo. A sub-chronic dietary study, preceded by 4-week parental and in utero exposure phase, investigated general safety. Administration of pAOS did not affect parental health nor pup characteristics. No effects specific for acidic oligosaccharides were observed in the subsequent sub-chronic study. Slight diffuse hyperplasia of epithelial layer of the urinary bladder was noted to result from concurrently elevated urinary sodium, due to high sodium in pAOS, and elevated urinary pH. This phenomenon was confirmed in a mechanistic (sub-chronic) study. In contrast, in rats fed pAOS in combination with NH4Cl, an acidifying agent, the induced low urinary pH completely prevented the development of urothelial hyperplasia. Hyperplasia induced by this mechanism in rats is considered not relevant to man. Based on the current knowledge we consider pAOS safe for human consumption under its intended use. © 2009 Elsevier Inc.

Many epidemiological studies suggest that elevated plasma fibrinogen concentrations form one of the most important independent risk factors in blood for cardiovascular disease and particularly atherosclerosis in humans. To clarify the effect of genetic factors, diets and their interactions on plasma fibrinogen concentrations, we examined plasma fibrinogen levels in four strains of mice, which differ in their susceptibility to cholesterol-induced atherosclerosis. When maintained on basal diet, two strains 129/J and C3H/HeJ exhibited a significantly higher plasma fibrinogen concentration (2.1 and 1.9 mg/ml) than C57BL/6J and BALB/C strains (1.5 and 1.4 mg/ml). The strongest and most rapid (1 week) increase of plasma fibrinogen (by all semi-synthetic diets) is observed in C57BL/6J mice, which are known to be highly susceptible to diet-induced atherosclerosis. After a period of 8 weeks an increase in plasma fibrinogen of approximately 30-50% was observed in all strains on all semi-synthetic diets. Remarkably, no increase was observed in the fibrinogen Aα- Bβ- and γ-chain mRNA levels in the liver on the same diets. These mRNA levels were even decreased by approximately 20-50% in all strains on an extremely atherogenic diet. It was found that: genetic background determines the plasma fibrinogen levels on basal diet; plasma fibrinogen levels are altered by diet; the extent of these changes depends on the genetic background: surprisingly, this increase of fibrinogen in plasma is independent of transcription; the diet-induced increase of fibrinogen was very fast in the very highly atherosclerosis-susceptible strain C57BL/6J having a low basal fibrinogen level, and very slow in the atherosclerosis-resistant strain C3H/HeJ having a high basal fibrinogen level. It might be concluded that it is the kinetics of the response of fibrinogen to diet rather than the actual level, which relates to atherosclerosis susceptibility. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Treatment of mice with high doses of endotoxin (ET) affected the CFU(s) population in bone marrow, spleen and liver. By 7 days after injection the number of CFU(s) in bone marrow had decreased and the number in spleen and liver had increased. The experimental data show that if mice were irradiated and reconstituted with bone marrow 7 days after ET treatment, the lodging pattern of injected cells followed qualitatively the same changes as the changes in numbers of CFU(s) present before irradiation in these organs. However, elevated numbers of CFU(s) cannot be maintained in the liver and can only partly be maintained in the spleen. In retransplantation experiments, CFU(s) from bone marrow and from bone marrow or liver after treatment of mice with ET have the same seeding efficiency in the spleen, indicating that there is no preferential homing of subclasses of CFU(s) in bone marrow or liver. It is concluded that the presence of CFU(s) and hemopoietic tissue may be an expression of the capability of the tissues for the maintenance of lodging CFU, but the initial lodging in these organs is also affected by other trapping mechanisms.

To study the possible reduction by eugenol of the mutagenicity and genotoxicity of benzoja]pyrene (B[a]P) in vivo, the X-lacZ-transgenic mouse strain 40.6 (Muta(TM)Mouse) was used. Male mice were fed a diet containing 0.4% (w/w) eugenol or a control diet for 58 days. On day 10, half of the mice received an i.p. dose of 100 mg/kg b.w. B[a]P. The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage, and expressed in E. coli C lacZ- recA-galE- bacteria. In both control mice and mice fed the eugenol diet, B[a]P treatment resulted in a similar, significant increase in lacZ mutant frequency. Eugenol was not mutagenic by itself By 32P-postlabelling analysis of the liver DNA using an analysis method with chromatographic conditions for B[a]P-DNA adducts, no effect of eugenol on the formation of B[a]P-DNA adducts in the λ-lacZ-transgenic mouse was found. By 32P-postlabelling analysis using an alkenylbenzene solvent system the amount of B[a]P-DNA adducts was lower in mice fed the eugenol diet than in mice fed the control diet but the decrease was not statistically significant. However, one spot indicative of an eugenol-associated DNA adduct was detected. The present data provide no evidence for antimutagenic or antigenotoxic potential of eugenol in vivo Furthermore, they suggest genotoxicity in vivo of eugenol per se.

Gram-negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high-density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer-membrane component of gram-negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose-dependently increased the early inflammatory response to Klebsiella pneumoniae-induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis. Chemicals / CAS: Apolipoprotein C-I; Apolipoproteins C; Lipopolysaccharides

Rats and mice are the most commonly used species as laboratory animal models of diseases in biomedical research. Environmental factors such as cage size, number of cage mates and cage structure such as environmental enrichment can affect the physiology and behavioural development of laboratory animals and their well-being throughout their lives. Therefore compromising the animals' well-being due to inadequate environmental conditions would diminish the value of the research models. In order to improve laboratory animals' well-being and promote the quality of animal based biomedical research, it is fundamentally important that the environment of the animals meets the animals' species typical behavioural needs. Standardisation of environmental enrichment for laboratory rats and mice therefore should provide possibilities for the animals to engage in at least the essential behavioural needs such as social contact, nest building, exploring and foraging. There is a wide variety of environmental enrichment items commercially available for laboratory mice and rats. However, how these items are used by the animals, their practicality in the laboratory and whether these enrichments might lead to increased variation in experimental results have not been widely assessed. In this study, we implemented two standardised enrichment items (shelters, nesting materials) for rats and mice at different animal units. We instructed the animal care staff in monitoring the use of enrichment items by the animals by means of a daily score sheet system. The animal staff's viewpoint on practicality of the standardised enrichment program was assessed with a monthly score sheet survey. Also we assessed whether the enriched environment affected breeding results and contributed to an increase in variation of experimental data from several participating current studies. Our results show that the animals readily used the provided enrichment items. A slight increase in workload for the animal staff was reported. However, the overall judgement was mainly reported as good. Breeding results and variation in experimental data did not reveal differences as compared to data from previous housing and/or non enriched housing conditions. Overall, the results indicate that standard environmental enrichment that is species appropriate may enhance the animal's well-being without undesirable side effects on the experimental outcome and daily working routine of the animal care staff.

Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen cells, and (b) lacZ and Dlb-1 in small intestine from λlacZ(+/0)/Dlb-1(a/b) mice. Induction of mutations by ethyl- and methylnitrosourea (ENU, MNU) and ethyl methanesulphonate (EMS) was investigated at 7 weeks after a single i.p. dose of each of these chemicals. In the small intestine, treatment with various dosages of ENU (10-150 mg/kg) resulted in a linear dose-response in both lacZ and Dlb-1. MNU (30 mg/kg) was also mutagenic in lacZ and Dlb-1, while EMS (250 mg/kg) did not significantly induce mutations in either gene. In spleen, ENU gave a linear dose-related response in both lacZ and hprt, MNU induced mutations in both lacZ and hprt, and EMS was only positive for lacZ. No differences in response were observed between single and split-dose treatment with ENU (1x50 or 5x10 mg/kg with a 1- or 7-day interval), both in spleen and small intestine, except for lacZ in small intestine, where the single high dose gave a significantly higher induction than the split dose with the 7-day interval. The overall results suggest that mutagenic effects of fractionated doses are generally additive. In most cases, the induction factor (ratio treated over controls) for mutations in lacZ was lower than that for hprt and Dlb-1, presumably due to a higher background in lacZ and/or a lower mutability of lacZ. The general concordance between the data for lacZ and the endogenous genes indicates that λlacZ transgenic mice are a suitable model to study induction of gene mutations in vivo. Chemicals/CAS: Alkylating Agents; Ethyl Methanesulfonate, 62-50-0; Ethylnitrosourea, 759-73-9; Genetic Markers; Hypoxanthine Phosphoribosyltransferase, EC 2.4.2.8; Methylnitrosourea, 684-93-5; Mutagens

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and β-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: glucose, 50-99-7, 84778-64-3; insulin, 9004-10-8; Blood Glucose; Insulin, 11061-68-0; RNA, Messenger; Triglycerides

Transgenic mice expressing human cholesteryl ester transfer protein (HuCETPTg mice) were crossed with apolipoprotein CI-knocked out (apoCI-KO) mice. Although total cholesterol levels tended to be reduced as the result of CETP expression in HuCETPTg heterozygotes compared with C57BL6 control mice (-13%, not significant), a more pronounced decrease (-28%, p < 0.05) was observed when human CETP was expressed in an apoCI-deficient background (HuCETPTg/apoCI-KO mice). Gel permeation chromatography analysis revealed a significant, 6.1-fold rise (p < 0.05) in the cholesteryl ester content of very low density lipoproteins in HuCETPTg/apoCI-KO mice compared with control mice, whereas the 2.7-fold increase in HuCETPTg mice did not reach the significance level in these experiments. Approximately 50% decreases in the cholesteryl ester content and cholesteryl ester to triglyceride ratio of high density lipoproteins (HDL) were observed in HuCETPTg/apoCI-KO mice compared with controls (p < 0.05 in both cases), with intermediate -20% changes in HuCETPTg mice. The cholesteryl ester depletion of HDL was accompanied with a significant reduction in their mean apparent diameter (8.68 ± 0.04 nm in HuCETPTg/apoCI-KO mice versus 8.83 ± 0.02 nm in control mice; p < 0.05), again with intermediate values in HuCETPTg mice (8.77 ± 0.04 nm). In vitro purified apoCI was able to inhibit cholesteryl ester exchange when added to either total plasma or reconstituted HDL-free mixtures, and coincidently, the specific activity of CETP was significantly increased in the apoCI-deficient state (173 ± 75 pmol/μg/h in HuCETPTg/apoCI-KO mice versus 72 ± 19 pmol/μg/h in HuCETPTg, p < 0.05). Finally, HDL from apoCI-KO mice were shown to interact more readily with purified CETP than control HDL that differ only by their apoCI content. Overall, the present observations provide direct support for a potent specific inhibition of CETP by plasma apoCI in vivo. Chemicals/CAS: Apolipoprotein C-I; Apolipoproteins C; Carrier Proteins; CETP protein, human; Cholesterol Ester Transfer Proteins; Glycoproteins; Lipoproteins

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, λlacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC → AT transitions, consistent with the O6-EtG lesion, and 28% TA → AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC → AT mutations and 22% were TA → AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Objective: The liver is the key organ involved in systemic inflammation, but the relation between hepatic inflammation and atherogenesis is poorly understood. Since nuclear factor-κB (NF-κB) is a central regulator of inflammatory processes, we hypothesized that chronically enhanced hepatic NF-κB activation, through hepatocyte-specific expression of IκB kinase-β (IKKβ) (LIKK), will aggravate atherosclerosis development in APOE*3-Leiden (E3L) mice. Methods and results: E3L.LIKK and E3L control littermates were fed a Western-type diet for 24 weeks. E3L.LIKK mice showed a 2.3-fold increased atherosclerotic lesion area and more advanced atherosclerosis in the aortic root with less segments without atherosclerotic lesions (11% vs. 42%), and more segments with mild (63% vs. 44%) and severe (26% vs. 14%) lesions. Expression of LIKK did not affect basal levels of inflammatory parameters, but plasma cytokine levels tended to be higher in E3L.LIKK mice after lipopolysaccharide (LPS) administration. E3L.LIKK mice showed transiently increased plasma cholesterol levels, confined to (V)LDL. This transient character resulted in a mild (+17%) increased cumulative plasma cholesterol exposure. Conclusion: We conclude that selective activation of NF-κB in hepatocytes considerably promotes atherosclerosis development which is (at least partly) explained by an increased sensitivity to proinflammatory triggers and transiently increased plasma cholesterol levels. © 2011 Elsevier Ireland Ltd.

Purpose: To identify the risk factors for the onset of arm-wrist-hand and neck-shoulder symptoms among office workers and to estimate the relative contribution of these risk factors by calculating Population Attributable Fractions (PAFs). Methods: A prospective cohort study was conducted among 1951 office workers with a follow-up duration of 2 years. Data on self-reported risk factors were collected at baseline and after 1 year of follow-up. Every 3 months, the occurrence of upper extremity symptoms was assessed using questionnaires. PAFs for individual risk factors were estimated based on Rate ratios (RRs) obtained from Poisson regression using Generalized Estimation Equations. Results: Previous disabling symptoms were identified as the most important risk factor for the onset of arm-wrist-hand and neck-shoulder symptoms. Modifiable risk factors for arm-wrist-hand symptoms with relatively large PAFs were: at least 4 h per day of self-reported computer use at work, high level of overcommitment, and low task variation and for neck-shoulder symptoms: supporting the arms during keyboard use and at least 4 h per day of self-reported mouse use at work. Compared to having 0 or 1 risk factor, the RR for arm-wrist-hand symptoms increased to 6.2 (95% CI 3.7-10.5) for having 5-7 potentially modifiable risk factors and for neck-shoulder symptoms to 3.0 (95% CI 2.1-4.4) for having 4 or 5 potentially modifiable risk factors. Conclusion: Preventive interventions at the population level should be aimed at changing modifiable risk factors with large PAFs. At the individual level, preventive interventions should be aimed at changing multiple modifiable risk factors simultaneously. © Springer-Verlag 2011.

Objective Epidemiologic studies on physical exposure during computer use have mainly focused on average exposure duration. In this study, we aimed to relate periods of high peak exposure during computer use with the occurrence of neck-shoulder (NS) and arm-wrist-hand (AWH) symptoms. Methods A prospective cohort study among 1951 office workers was carried out for two years, with periodical questionnaires and continuous measurements of computer input use. To define peak exposure, a distinction was made between peak days and weeks. Peak days were defined as days with a long duration of computer (ie, ≥4 hours) or mouse use (ie, ≥2.5 hours) or days with high frequency of mouse (ie, ≥20 clicks per minute) or keyboard use (ie, ≥160 keystrokes per minute). Weeks containing ≥3 peak days were considered peak weeks. Independent variables were numbers of peak days and peak weeks during a 3-month measurement period; dependent variables were self-reported NS and AWH symptoms during the following 3-month measurement period. Results Valid data were available for 2116 measurements of 774 office workers. No relation was found between any of the peak exposure parameters and AWH symptoms or with peak exposure in duration and NS symptoms. Most parameters referring to high frequency-related peak exposure were associated with less NS symptoms, but the effect estimates were very small and the confidence intervals close to the null. Conclusion In this study, we found no indication that high peaks in computer use were related to the occurrence of NS or AWH symptoms. This work is licensed under a Creative Commons Attribution 4.0 International License.

Dyslipidemia and inflammation are well known causal risk factors the development of atherosclerosis. The interplay between lipid metabolism and inflammation at multiple levels in metabolic active tissues may exacerbate the development of atherosclerosis, and will be discussed in this review. Cholesterol, fatty acids and modified lipids can directly activate inflammatory pathways. In addition, circulating (modified) lipoproteins modulate the activity of leukocytes. Vice versa, proinflammatory signaling (i.e. cytokines) in pre-clinical models directly affects lipid metabolism. Whereas the main lipid-lowering drugs all have potent anti-inflammatory actions, the lipid-modulating actions of anti-inflammatory agents appear to be less straightforward. The latter have mainly been evaluated in pre-clinical models and in patients with chronic inflammatory diseases, which will be discussed. The clinical trials that are currently conducted to evaluate the efficacy of anti-inflammatory agents in the treatment of cardiovascular diseases may additionally reveal potential (beneficial) effects of these therapeutics on lipid metabolism in the general population at risk for CVD. © 2013 Elsevier Ireland Ltd.