Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium

The rat theta class glutathione S-transferase (GST) 5-5 has been shown to affect the mutagenicity of halogenated alkanes and epoxides. In Salmonella typhimurium TA1535 expressing the rat GST5-5 the number of revertants was increased compared to the control strain by CH2Br2, ethylene dibromide (EDB) and 1,2,3,4-diepoxybutane (BDE); in contrast, mutagenicity of 1,2-epoxy-3-(4'-nitrophenoxy)propane (EPNP) was reduced. S.typhimurium TA1535 cells were transformed with an expression plasmid carrying the cDNA of the human theta ortholog GST1-1 either in sense or antisense orientation, the latter being the control. These transformed bacteria were utilized for mutagenicity assays. Mutagenicity of EDB, BDE, CH2Br2, epibromohydrin and 1,3-dichloroacetone was higher in the S.typhimurium TA1535 expressing GSTT1-1 than in the control strain. The expression of active enzyme did not affect the mutagenicity of 1,2-epoxy-3-butene or propylene oxide, GSTT1-1 expression reduced the mutagenicity of EPNP. Glutathione S-transferase 5-5 and GSTT1-1 modulate genotoxicity of several industrially important chemicals in the same way. Polymorphism of the GSTT1 locus in humans may therefore cause differences in cancer susceptibility between the two phenotypes.

Soft tissue human thigh and buttock finite element model to simulate vehicle seat cushion indentation

Accurate modelling of automotive occupant posture is strongly related to the mechanical interaction between human body soft tissue and flexible seat components. This paper presents a finite-element study simulating the deflection of seat cushion foam and supportive seat structures, as well as human buttock and thigh soft tissue when seated. The thigh-buttock surface shell model was based on 95th percentile male subject scan data and made of two layers, covering thin to moderate thigh and buttock proportions. To replicate the effects of skin and fat, the neoprene rubber layer was modelled as a hyperelastic material with viscoelastic behaviour. The analytical seat model is based on a Ford production seat. The result of the finite-element indentation simulation is compared to a previous simulation of an indentation with a hard shell human model of equal geometry, and to the physical indentation result. We conclude that SAE composite buttock form and human-seat indentation of a suspended seat cushion can be validly simulated.

Numerical optimization of soft-mold aided co-curing process of advanced grid-stiffened composite structures

The co-curing process for advanced grid-stiffened (AGS) composite structure is a promising manufacturing process, which could reduce the manufacturing cost, augment the advantages and improve the performance of AGS composite structure. An improved method named soft-mold aided co-curing process which replaces the expansion molds by a whole rubber mold is adopted in this paper. This co-curing process is capable to co-cure a typical AGS composite structure with the manufacturer’s recommended cure cycle (MRCC). Numerical models are developed to evaluate the variation of temperature and the degree of cure in AGS composite structure during the soft-mold aided co-curing process. The simulation results were validated by experimental results obtained from embedded temperature sensors. Based on the validated modeling framework, the cycle of cure can be optimized by reducing more than half the time of MRCC while obtaining a reliable degree of cure. The shape and size effects of AGS composite structure on the distribution of temperature and degree of cure are also investigated to provide insights for the optimization of soft-mold aided co-curing process.

Combining the diaza-Diels-Alder reaction and palladium-catalyzed aminations to prepare amino-substituted porphyrins

A strategy to tackle the synthesis of azoporphyrins with unsubstituted terminal meso positions was investigated. It comprised the combination of diaza-Diels–Alder (DADA) reaction of 1,3-dienes with dialkyl azodicarboxylates, decarboxylative hydrolysis of the bis(carbamates), palladium-catalyzed amination of bromoporphyrin precursors, and retro-DADA reactions to release the ultimate targets. The somewhat confused historical results on the DADA reactions of 1,3-cyclohexadiene were clarified, but the hydrolyses yielded extremely air-sensitive amines which decomposed completely in minutes via autooxidation and retro-DADA reaction. With anthracene or 2,3-dimethyl-1,3-butadiene as the diene, the synthesis of azoporphyrin was not achieved but three amino-substituted porphyrins were obtained in moderate yields under mild conditions. The X-ray crystal structures of several of the intermediates and the final aminoanthracene-porphyrin nickel(II) complex were determined.

A new method for the in vivo identification of mechanical properties in arteries from cine MRI images: Theoretical framework and validation

Quantifying the stiffness properties of soft tissues is essential for the diagnosis of many cardiovascular diseases such as atherosclerosis. In these pathologies it is widely agreed that the arterial wall stiffness is an indicator of vulnerability. The present paper focuses on the carotid artery and proposes a new inversion methodology for deriving the stiffness properties of the wall from cine-MRI (magnetic resonance imaging) data. We address this problem by setting-up a cost function defined as the distance between the modeled pixel signals and the measured ones. Minimizing this cost function yields the unknown stiffness properties of both the arterial wall and the surrounding tissues. The sensitivity of the identified properties to various sources of uncertainty is studied. Validation of the method is performed on a rubber phantom. The elastic modulus identified using the developed methodology lies within a mean error of 9.6%. It is then applied to two young healthy subjects as a proof of practical feasibility, with identified values of 625 kPa and 587 kPa for one of the carotid of each subject.

Experimental measurement of the mechanical properties of carotid atherothrombotic plaque fibrous cap

Eleven carotid atherothrombotic plaque samples were harvested from patients. Three samples that were highly calcified were discarded, while eight yielded results. The elastic properties of the material were estimated by fitting the measured indentation response to finite element simulations. The methodology was refined and its accuracy quantified using a synthetic rubber. The neo-Hookean form of the material model gave a good fit to the measured response of the tissue. The inferred shear modulus μ was found to be in the range 7-100 kPa, with a median value of 11 kPa. A review of published materials data showed a wide range of material properties for human atherothrombotic tissue. The effects of anisotropy and time dependency in these published results were highlighted. The present measurements were comparable to the static radial compression tests of Lee et al, 1991 [Structure-dependent dynamic behaviour of fibrous caps from human atherosclerotic plaques. Circulation 83, 1764-1770].

In vivo non-invasive high resolution MR-based method for the determination of the elastic modulus of arterial vessels

The mechanical properties of arterial walls have long been recognized to play an essential role in the development and progression of cardiovascular disease (CVD). Early detection of variations in the elastic modulus of arteries would help in monitoring patients at high cardiovascular risk stratifying them according to risk. An in vivo, non-invasive, high resolution MR-phase-contrast based method for the estimation of the time-dependent elastic modulus of healthy arteries was developed, validated in vitro by means of a thin walled silicon rubber tube integrated into an existing MR-compatible flow simulator and used on healthy volunteers. A comparison of the elastic modulus of the silicon tube measured from the MRI-based technique with direct measurements confirmed the method's capability. The repeatability of the method was assessed. Viscoelastic and inertial effects characterizing the dynamic response of arteries in vivo emerged from the comparison of the pressure waveform and the area variation curve over a period. For all the volunteers who took part in the study the elastic modulus was found to be in the range 50-250 kPa, to increase during the rising part of the cycle, and to decrease with decreasing pressure during the downstroke of systole and subsequent diastole.

Low carbon fuels and commodity chemicals from waste gases – systematic approach to understand energy metabolism in a model acetogen

Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains.