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SRAM-based Field Programmable Gate Arrays (SRAM-FPGA) are more and more employed in today's applications. In space and avionic applications their operations might be harmed by occurrence of radiation-induced upsets, or Single Event Upsets (SEU), which require the adoption of mitigation techniques. In these devices the majority of the configuration memory rules the interconnection setting. In devices employing "switch matrix" routing, the density of interconnections in switch arrays seems to be a critical point. The higher the interconnection density (i.e., the higher the number of interconnection segments activated by the same switch matrix), the higher the probability of an upset due to a configuration bit controlling the switch matrix. This paper presents an approach to estimate the SEU sensitivity of programmable interconnections of SRAM-based FPGAs as a function of the density of programmable interconnection points inside device configurable logic blocks. A probabilistic model of the SEU effects in programmable interconnection points of Xilinx SRAM-FPGAs is described. The application of the proposed approach to a set of sample designs is illustrated.

Nowadays the reliability issues of SRAM-based Field Programmable Gate Arrays (FPGAs) operating in harsh environments are well understood. One major effect is Single Event Upsets (SEUs), which are able to invert the stored logical value in flip-flops and memory cells. This issue is more serious when the affected memory cells are part of the configuration memory used for programming the circuit functionality. The consequences may be alterations of the circuit functionality causing errors which may only be corrected by reprogramming the device. For a better understanding of the robustness of programmed circuits, this paper compares two decoders for Error Correction Codes (ECCs). A Hamming Decoder and a One-Step Majority Logic Decoder (OS-MLD) for the Difference-Set Cyclic Codes (DSCC) are analyzed yielding surprisingly unexpected results for their SEU susceptibility, which are interesting for application designers.

Reliability is a major concern for electronic circuits, especially for those that operate in harsh environments. One source of problems are Single Event Upsets (SEU), which change the value of flip flops and memory cells. SEUs are a major issue for SRAM-based Field Programmable Gate Arrays (FPGAs), as they may alter the circuit functionality, creating errors that will only be removed if the device is reprogrammed. The cost of traditional techniques to deal with SEUs, like triplication, can be excessive in some applications. One example are Space systems, in which power consumption and weight are limited. In those cases, the use of ad hoc protection techniques that can reduce the cost is interesting. In this paper, new protection techniques for adaptive equalizers implemented in SRAM-based FPGAs are presented. The proposed techniques use the knowledge of the equalizer to provide effective protection at a lower cost. The results show a reduction of up to 70% in the use of resources in comparison to the commercial XTMR solution.

The paper discusses the experimental validation of fault injection analyses performed with the FLIPPER tool. Failure probabilities obtained by fault injection were compared against failure probabilities obtained at accelerated proton testing of a benchmark design provided by the European Space Agency.

The paper describes how FLIPPER can be used to analyze the SEU sensitivity of designs implemented by means of SRAM-based FPGAs by both fault injection and radiation testing. The comparison between fault injection experiment and proton testing on selected designs is presented in the paper. The excellent agreement represents a significant achievement toward the FLIPPER validation.

Commercial-off-the-shelf devices are often advocated as the only solution to the increasing performance requirements in space applications. This paper presents the solutions developed in the frame of the European Space Agency's HiRel program, concluded in December 2014, where a number of techniques proposed in the past 10 years have been used to design a highly reliable system, which has been selected for forthcoming space missions. This paper presents the system architecture, describes performed evaluations, and discusses the results.

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the break-down of phospholipid-bound polyunsaturated fatty acids of cellular membranes. Despite its biological relevance, this aldehyde is only occasionally determined due to the complexity of previously described procedures. Here we present a simple and very sensitive method for the detection of HNE in biological samples. The method is based on the measurement of the 2,4-dinitrophenylhydrazone (DNPH) of the aldehyde by electrochemical detection after separation by reverse-phase high-performance liquid chromatography (HPLC). The greater sensitivity of this procedure as compared to the ultraviolet detection method commonly employed to measure DNPH derivatives of aldehydes after HPLC will allow the detection of HNE below the pmol level. The detection of HNE is highly reproducible even in normal tissues and cells. Increased amounts of HNE were detected in the livers of animals intoxicated with prooxidant agents such as carbon tetrachloride, bromotrichloromethane or bromobenzene. An exponential increase in HNE (and in malondialdehyde) was measured in peroxidizing liver microsomes (in the NADPH/Fe-dependent system). The method is also suitable for the study of very small samples, since HNE could be detected in approximately 1 million cultured cells (polyoma virus-transformed baby hamster kidney fibroblasts); the level rose after exposure of the cells to a Fe3+/ADP prooxidant system.

The paper describes the SIDERALE experiment that was hosted as a piggy back payload on SoRa LDB (Sounding Radar Long Distance Balloon) mission by the Italian Space Agency (ASI). SIDERALE was aimed at testing a detector for high energy astrophysics applications based on a 4x4 pixel CZT solid state sensor. An onboard data handling computer, a mass memory and a power supply units were integrated in SIDERALE. Furthermore an innovative telemetry system BIT (Bi-directional Iridium Telemetry) was used in order for SIDERALE to be autonomous and independent from the hosting payload. In the paper a preliminary analysis of flight and scientific data is discussed.

Boradband, single pole four throw (SP4T) switches have been manufactured in MEMS technology and tested up to 40 GHz.

We report on the development of a 3D position sensitive prototype suitable as focal plane detector for Laue lens telescope. The basic sensitive unit is a drift strip detector based on a CZT crystal, (similar to 19x8 mm(2) area, 2.4 mm thick), irradiated transversally to the electric field direction. The anode side is segmented in 64 strips, that divide the crystal in 8 independent sensor (pixel), each composed by one collecting strip and 7 (one in common) adjacent drift strips. The drift strips are biased by a voltage divider, whereas the anode strips are held at ground. Furthermore, the cathode is divided in 4 horizontal strips for the reconstruction of the third interaction position coordinate. The 3D prototype will be made by packing 8 linear modules, each composed by one basic sensitive unit, bonded on a ceramic layer. The linear modules readout is provided by a custom front end electronics implementing a set of three RENA-3 for a total of 128 channels. The front-end electronics and the operating logics (in particular coincidence logics for polarisation measurements) are handled by a versatile and modular multi-parametric back end electronics developed using FPGA technology.

Recent progress in the CdZnTe (CZT) based detectors prototypes suggests the possibility of an efficient use as 3D detectors. In this paper we discuss how the z coordinate of the interacting photon can be obtained in thick pixellated CZT crystals with a spatial resolution equal to the linear dimension of the implemented pixel (mm). Two different approaches will be described. The first considers a fine segmentation of multi anode and cathode electrodes arranged in orthogonal strips and biased with an electric field transversal to the photons arrival line (PTF = Planar Transversal Field): this solution tends to simplify the readout electronics although at the expense of some complexity in detectors assembling. The second one is based on a more classical configuration, single cathode and multi electrodes anode (exploiting the small pixel effect) biased with a field parallel to the impinging photons direction (PPF = Parallel Transversal Field). This approach needs a more complex electronics to analyze the signal shape of each interaction in order to evaluate, among other parameters, the interaction depth. Conversely in this configuration the focal plane can be assembled with relative simplicity. Both the configurations can be disposed in a stack to improve the working energy range through an effective use of the Compton effect.

The comparison between FLIPPER fault injection experiment and radiation ground-testing on selected SRAM-FPGA designs is presented in the paper. The excellent agreement represents a significant achievement toward the validation of the FLIPPER platform.

We describe a methodology for analyzing the robustness of circuits implemented by SRAM-based FPGAs against the accumulation of soft errors within the configuration memory. A detailed analysis of the fault injection data is presented.

In the last years MEMS technology has demonstrated to be a very attractive solution for the production of high performances RF switches. As such, it is an enabling technology for the development of high performances, high complexity and very compact switch matrices, able to cope with the ever increasing requirements in terms of capability, flexibility and connectivity of modern telecom payloads. In order to achieve this goal, integration and reliability issues linked to that technology and to that kind of units shall be extensively explored since R&D efforts have been until now mainly focused at the component level. In this frame Thales Alenia Space Italia is involved in several R&D programs aimed at the development and reliability investigation of RF MEMS based switch matrices. These include the ESA programs "Very Large Order Switch Matrices Using MEMS Technology" and "High Reliability MEMS Redundancy Switch", the development of a MEMS switch matrix unit to be included in Alphasat experimental payload, the development of in-house foundry capabilities for MEMS on ceramics and MEMS integrated on ISP (Integrated Substrate Package). This paper reports about the studies conducted in the frame of these activities and their main findings. One of the main challenges in manufacturing very compact, high order switching matrices is the need to route an high number of RF and DC signals in a small space while assuring an high isolation between channels and signal integrity on control lines. Integration and packaging thus play a key role. In the paper several switch matrix architectures are presented along with different integration and packaging solutions based on LTCC (Low Temperature Co-fired Ceramics) technology. A trade off analysis among size/weight, RF-performances, modularity, cost, complexity and reliability is performed. Integration of such equipments inside existing and next generation telecom payloads is addressed as well. RF-MEMS technology reliability is still an open point which has prevented until now its use on-board spacecrafts. In this paper different MEMS-based switching cells topologies and technologies are analyzed in terms of RF-performances, reliability and ease of integration. Their use as switching units inside complex switching networks is described, along with the related reliability and integration issues. Design solutions aimed at improving the switching cell long term reliability are addressed as well. Finally a tentative qualification plan for RF MEMS switch based units is presented.

The aim of this study was to study ultrastructural changes and lipid peroxidation in rabbits lung after massive complement activation and leukocyte aggregation. A prolonged and massive leukocyte activation was induced by intraperitoneal inoculation of zymosan suspected in paraffin. Fifteen animals (group 3) were given 0.6 g/kg of zymosan, 22 animals (group 2) received 1 g/kg, and 11 rabbits (group 1) were treated with paraffin alone and served as controls. An acute mortality rate of 40% was observed in group 3 and of 68% in group 2. Surviving animals were studied for 10 days. In these animals a marked decrease in circulating granulocytes and a progressive decline in arterial PO2 were recorded (PO2 on day 10 in group 2 animals was 51.94 +/- 4.26, p less than 0.01). Microscopic and ultrastructural evaluation revealed sequestration of granulocytes in the pulmonary microvasculature. Studies of lung homogenates demonstrated increased levels of lipid peroxide derivative malondialdehyde (group 2 rabbits, 1624 +/- 638; group 1, 795 +/- 57 pm/mg pt, p less than .001) and decreased levels of the tissue antioxidant alpha-tocopherol. The results of this study are compatible with the hypothesis of leukocyte-mediated injury through production of oxygen radicals.

In the framework of an on-going R&D program on CZT detectors for astrophysical applications, we have started a study to address peculiar problems affecting this kind of detectors (e.g. response dependent on the interaction depth and multiple hit events) using a digital approach: the output of an analog front end chain based on a fast charge preamplifier, which preserves the shape of the signal as generated by the detector, sampled with a fast ADC, is processed to extract information to improve the performance, in particular the energy resolution. In this context, we have developed a flexible readout electronics to be used to characterize CZT array and test digital processing algorithms. The system includes analog front-end electronics, digital signal processing based on an FPGA board, and a Gb Ethernet interface for PC-based data acquisition. Current scheme allows the readout of a 4x4 pixels, which allows us to test the algorithms even for multi-pixels events and checking cross-talk effects. However, the system has been designed to be easily extended to an higher number of pixels by duplicating the Analog Front End board. Up to 4 AFEE boards can be managed by the system (thus supporting 64 channels, allowing a 16x16 array to be readout). Performance of the system with a commercial 4x4 pixels CZT (eV-products) will be reported in the paper.

In the context of R&D studies financed by the Italian Space Agency (ASI), a feasibility study to evaluate the Italian Industry interest in medium-large scale production of enhanced CZT detectors has been performed by an Italian Consortium. The R&D investment aims at providing in-house source of high quality solid state spectrometers for Space Astrophysics applications. As a possible spin-off industrial applications to Gamma-ray devices for non-destructive inspections in medical, commercial and security fields have been considered by ASI. The short term programme mainly consists of developing proprietary procedures for 2-3" CZT crystals growth, including bonding and contact philosophy, and a newly designed low-power electronics readout chain. The prototype design and breadboarding is based on a fast signal AD conversion with the target in order to perform a new run for an already existing low-power (

As SRAM-based field-programmable gate arrays (FPGAs) are introduced in safety- or mission-critical applications, the availability of suitable Electronic Design Automation (EDA) tools for predicting systems dependability becomes mandatory for designers. Nowadays designers can opt either for workload-independent EDA tools, which provide information about system's dependability disregarding the workload the system is supposed to elaborate when deployed in the mission, or workload-dependent approaches. In this paper, we compare two tools for predicting the effects of soft errors in circuits implemented using SRAM-based FPGAs, a workload-independent one (STAR) and a workload-dependent one (FLIPPER). Experimental results show that the two tools are complementary and can be used fruitfully for obtaining accurate predictions.

SRAM based reprogrammable FPGAs are sensitive to radiation-induced single event upsets (SEU), not only in their user flip-flops and memory, but also in the configuration memory. Appropriate mitigation has to be applied if they are used in space, for example the XTMR scheme implemented by the Xilinx TMRTool and configuration scrubbing. The FLIPPER fault injection platform, described in this paper, allows testing the efficiency of the SEU mitigation scheme. FLIPPER emulates SEU-like faults by doing partial reconfiguration and then applies stimuli derived from HDL simulation (VHDL/Verilog test-bench), while comparing the outputs with the golden pattern, also derived from simulation. FLIPPER has its device-under-test (DUT) FPGA on a mezzanine board, allowing an easy exchange of the DUT device. Results from a test campaign are presented using a design from space application and applying various levels of TMR mitigation.

To evaluate the glucose metabolism in acromegalic patients before and after treatment with octreotide LAR.This was a prospective and longitudinal study involving 30 patients from the acromegaly research outpatient clinic of the Endocrinology unit of the HUCFF/UFRJ. They underwent clinical and laboratorial evaluations, with measurements of growth hormone (GH), insulin-like growth factor type I (IGF-I), insulin, proinsulin, C peptide, glycosylated hemoglobin (HbA1c), IGF binding protein type 1 (IGFBP-1) and glucose, during oral glucose tolerance test (OGTT), before and after six months of treatment with octreotide LAR. The Wilcoxon signed-rank test was used and values of 5% were considered statistically significant.We found 16 (54%) patients with normal glucose tolerance, 7 (23%) with impaired glucose tolerance and 7 (23%) diabetics. Twelve patients completed the six-month treatment, out of which three showed worsening of glucose tolerance and two (diabetics) had worse blood glucose control. Whereas there was an increase in waist circumference (p=0.03), there was a decrease in GH (p=0.04), with %IGF-I above the upper limit of reference values (% ULRV) [p=0.001], insulin (p=0.019), C peptide levels (p=0.002) and homeostatic model assessment (HOMA-IR) [p=0.039].In this series, treatment with octreotide LAR led to a worsening of glucose tolerance in three non-diabetic patients and worsened glycemic control in two diabetics, in spite of reducing insulin resistance.

This paper reports on the successive improvements introduced in the shunt switches fabricated with the RF MEMS multiuser technology platform available at FBK-irst. In the course of a multiyear development several technological features and design methods have been made available to enhance the operation of capacitive switches. This work analyzes their effects by reviewing the behaviour of the FBK-irst capacitive switches at three different stages of this optimization process. Improvements have been assessed by means of DC electromechanical characterizations, which use a simple quasistatic C-V measurement to extract the switch actuation voltage and the capacitance in the on and off states (Con and Coff) and RF measurements. The addition of a floating metal layer into the process flow has allowed a great increase of the switch on state capacitances, getting Con/Coff ratios of 200, up to 50 times greater than the ones obtained for the same structures without this feature.

Benefits resulting from the adoption of SRAM-based FPGAs as design target technology in space applications are manifold. These devices, however, exhibit a potentially high susceptibility to single event upsets (SEU) due to the presence of a large number of configuration memory cells. As fault injection alone is not able to reach every circuitry inside FPGA, radiation ground testing is mandatory in order to perform the analysis on a larger set of SEU upsets. This paper presents a radiation test methodology for Xilinx Virtex FPGAs based on the THESIC+ system.

Space research requires increasingly huge amounts of scientific data. Next generation satellites will have on-board supercomputing capabilities to perform efficient information processing and overcome the possible limit imposed by communication bandwidth to ground receiving stations. They will also have to survive even longer term missions; thus reliability and fault tolerance will be a major concern, to cope with radiation induced faults. Flexibility also emerges as a desirable requisite for on-board processing system to implement new functionalities and run different algorithms for the ongoing mission. The trend is towards multiprocessor architecture in which processing nodes and memories are connected through high bandwidth interconnection networks. The paper presents a fault-tolerance strategy for an FPGA implementation of a redundant multistage interconnection network (MIN), for a space multi-sensor system. The mechanism is endowed with fault diagnosis ability which allows one to exploit MIN intrinsic reconfiguration capabilities, as well as the reprogrammability of SRAM-based FPGAs.

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

1. The ability of ascorbic acid to protect from prooxidant-induced toxic injury was investigated in isolated, intact rat hepatocytes, whose ascorbic acid content had been restored by means of exogenous supplementation. 2. Ascorbate-supplemented and ascorbate-non-supplemented cells in suspension were treated with a series of different prooxidants (allyl alcohol, diethyl maleate, carbon tetrachloride, menadione), and the development of lipid peroxidation and cell injury was evaluated. 3. With allyl alcohol and diethyl maleate, ascorbic acid was able to protect cells from both lipid peroxidation and cell injury. The same protection was offered by ascorbate also in hepatocytes obtained from vitamin E-deficient animals. 4. With carbon tetrachloride, ascorbate supplementation did not affect the initial steps of lipid peroxidation, but nevertheless provided a marked protection against lipid peroxidation and cell injury at later times of incubation. The protection was unaffected by the vitamin E content of cells. 5. With menadione, a toxin which does not induce lipid peroxidation, ascorbic acid did not protect cells against injury. 6. It is concluded that ascorbic acid can act as an efficient antioxidant in isolated rat liver cells, with protection against cell injury. The antioxidant effect appears primarily to involve membrane lipids, and can be independent from the cellular content of vitamin E, thus suggesting that ascorbic acid can play a direct and independent role in the intact cell, in addition to its synergistic interaction with vitamin E described in other models.

A number of antioxidant systems protect cells against oxidative stress. Among these, ascorbic acid (AA) is generally believed to play an important role both in scavenging aqueous radicals and in regenerating vitamin E. The efficiency of AA as an antioxidant is readily apparent in in vitro systems in which isolated hepatocytes are incubated with prooxidants. We have shown, for instance, that high intracellular levels of AA afford a marked protection against allyl alcohol-induced lipid peroxidation and cell death, despite a quite similar glutathione (GSH) depletion (1). The antioxidant activity of AA results in AA consumption with formation of semidehydroascorbic acid radicals which, by dismutation, can produce dehydroascorbic acid (DHAA). The latter can be converted to degradation products, but can also bind to cellular macromolecules, thus producing toxic effects. Therefore, the cellular systems capable of reducing the oxidized forms of AA are important in eliminating such toxic effects and in restoring the antioxidant potential of AA. Evidence (2, 3) suggests that GSH is involved in the redox cycling of AA by rapidly reducing the oxidized forms of the latter. In fact the addition of DHAA to isolated hepatocytes results (1) in a marked increase of the intracellular content of AA at the expenses of GSH, which is oxidized to GSSG. Pretreatment of the hepatocytes with diethylmaleate or 1,3-bis(2-chloroethyl-1- nitrosourea) (BCNU) strongly decreases the capacity of the hepatocytes to reduce DHAA to AA.

1. The mechanisms of the liver damage produced by three glutathione (GSH)-depleting agents, bromobenzene, allyl alcohol and diethyl maleate, were investigated. 2. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. 3. Changes in antioxidant systems by alpha-tocopherol (vitamin E) and ascorbic acid were studied. A decrease in the hepatic level of vitamin E, and a change in the redox state of vitamin C (increase in oxidized over reduced form) were evident whenever extensive lipid peroxidation developed. However, in the case of bromobenzene intoxication these alterations preceded lipid peroxidation, and may be an index of oxidative stress leading to subsequent membrane damage. 4. Experiments carried out with vitamin E-deficient or supplemented diets indicated that pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E-deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. In animals fed a vitamin E-supplemented diet, bromobenzene and allyl alcohol had only limited toxicity, and diethyl maleate none, in spite of similar hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of toxicity by GSH-depleting agents.

Previous studies from our laboratory (1–3) have shown that the intoxication of mice with three prototypical glutathione (GSH) depleting agents (bromobenzene, diethylmaleate and allyl alcohol) is followed by the development of lipid peroxidation and liver necrosis after the hepatic GSH depletion has reached critical values. The treatment of the intoxicated animals with the antioxidants Trolox C or desferrioxamine, completely prevents both lipid peroxidation and liver necrosis, while not changing at all the extent of the covalent binding of bromobenzene metabolites to liver protein. In subsequent studies, the relationships among the various antioxidant systems (namely, vit. E, GSH and ascorbic acid) of the liver cell have been investigated under conditions of severe GSH depletion, like those induced by the GSH depleting agents mentioned above. Such an abrupt loss of the GSH antioxidant system could reasonably affect the other antioxidant systems. According to some authors (4,5), in fact, GSH is directly involved in the enzymatic system (“tocopheroxy radical reductase“) that reduces the oxidized form of tocopherol; on the other hand, according to others (6,7) the tocopherol regenerating system consists of ascorbic acid that is converted in the reaction to semidehydroascorbic acid radical and then to dehydroascorbic acid. Even in the latter case GSH may be involved in the reduction of the oxidized form of ascorbic acid (dehydroascorbic acid) (8,9). Alternatively, GSH depletion could affect the other antioxidant systems, by decreasing the disposition of hydrogen peroxide through GSH peroxidase. Vit. E may be consumed as a result of an increased formation of lipid peroxides in cell membranes, while ascorbate may be involved by direct interactions with oxy-radicals, especially with hydroxyl radicals.

Primary cultures of rat hepatocytes were used to explore the mechanisms of the toxicity of aryl halides. The sensitivity of the hepatocytes to chloro-, bromo-, and iodobenzene was enhanced by inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In each case, the increased cell killing depended on the metabolism of the toxicant, a result shown by the protective effect of SKF-525A, an inhibitor of mixed function oxidation. BCNU decreased the metabolism of [ 14 C]bromobenzene and the covalent binding of its metabolites by 20%. Chelation by deferoxamine of a cellular source of ferric iron prevented the cell killing in the presence or absence of BCNU. Deferoxamine had no effect on the metabolism or the covalent binding of [ 14 C]bromobenzene. Similarly, the antioxidant N,N′ -diphenyl- p -phenylenediamine (DPPD) reduced the cell killing and had no effect on the metabolism of [ 14 C]bromobenzene. Thus, the toxicity of the three aryl halides was manipulated in ways that modify the sensitivity of hepatocytes to an oxidative stress, and the changes in cell killing occurred without parallel changes in the metabolism of [ 14 C]bromobenzene or the covalent binding of its metabolites.

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by alpha-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents.

During the last decades it has been recognized (1–3) that peroxidation of cellular membranes is an important event in the pathogenetic mechanisms of the liver injury induced by chemicals, such as CC14 or BrCCl3 , which give, upon metabolism, reactive free radicals. The latter ones alkylate cellular macromolecules but do not induce glutathione (GSH) depletion. It was subsequently shown (4–9) that lipid peroxidation is also strictly associated with the liver necrosis induced by chemi cals, such as bromobenzene and acetaminophen, which are converted to electrophilic intermediates giving extensive GSH conjugation and consequent GSH depletion. We have studied in particular the liver injury producedin vivo by three prototypical GSH depleting agents which undergo different fates in the liver cell: i) bromobenzene, that is metabolized by the microsomal monooxygenase system with consumption of NADPH (10–12); ii) allyl alcohol that is metabolized by the cytosolic enzyme alcohol dehydrogenase to acrolein and by aldehyde dehydrogenase to acrylic acid, with production of NADH (13,14); iii) and diethylmaleate which is mainly conjugated with GSH by GSH-transferases without previous metabolism (15).

NMRI Albino mice, in which the hepatic glutathione (GSH) content was decreased by nearly 50% by either the administration of a pure glucose diet or by starvation, were intoxicated with aryl halides, bromobenzene, and iodobenzene (13 and 9 mmol/kg body weight, respectively, p.o.). After both intoxications, the hepatic glutathione content decreased rapidly to very low values, and liver necrosis, as assessed by serum transaminase levels, occurred in about 45 or 60% of the animals (in the case of bromobenzene or iodobenzene, respectively) after a lag phase of 9 or 6 hr. In both instances liver necrosis was evident only when the hepatic GSH depletion reached a threshold value (3.5-2.5 nmols/mg protein). The same threshold value was evident for the occurrence of lipid peroxidation (measured as both carbonyl functions and conjugated dienes in liver phospholipids). The possibility that the depletion in hepatic GSH level is capable of inducing lipid peroxidation and necrosis could be supported by the fact that similar results were obtained after the administration of diethylmaleate (12 mmol/kg, p.o.), a drug which is expected to conjugate directly with GSH without previous metabolism. The covalent binding of reactive metabolites to cellular macromolecules was determined in the case of bromobenzene poisoning. A dissociation between liver necrosis and covalent binding was observed in experiments in which Trolox C, a lower homolog of vitamin E, was administered (270 μmol/kg) 9 and 13 hr after bromobenzene poisoning. The treatment with Trolox C, in fact, almost completely prevented both liver necrosis and lipid peroxidation, while the extent of the covalent binding of bromobenzene metabolites to liver proteins was not altered.

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.

When liver microsomes were incubated with NADPH under anaerobic conditions either in the presence or in the absence of EDTA, no substantial amount of malonic dialdehyde (MDA) was formed and no substantial decrease in glucose-6-phosphatase (G-6-Pase) activity was seen. The addition of CCl4 to these in vitro systems had minor effects on both MDA production and the enzyme activity. When, on the contrary, the incubation was carried out under aerobic conditions in the absence of EDTA, a marked production of MDA occurred and G-6-Pase activity was almost completely destroyed. These results confirm the findings of Glende et al. [(1976) Biochem. Pharmacol.25, 2163–2170]. The presence of EDTA in the aerobic system reduced both the extent of lipoperoxidation and the decline in enzyme activity. The addition of CCl4 to these systems resulted in some increase in both MDA formation and G-6-Pase inactivation. The relationships between various steps of the lipid peroxidation process and the inactivation of G-6-Pase were then studied. It was observed that when the incubation was carried out under anaerobic conditions with carbon tetrachloride, the binding of CCl4 free radicals to microsomal lipids (as evidenced by the g.l.c. analysis of fatty acid methyl esters with an electron-capture detector, ECD) was qualitatively identical to that observed after CCl4 poisoning in vivo. Also, the diene conjugation absorption detected in the samples incubated in vitro was similar to that observed in the in vivo situation. Furthermore, when the fatty acid methyl esters of the lipids of liver microsomes incubated as above were analyzed by thin-layer chromatography, a typical spot (“D” spot), previously reported to occur in the fatty acid methyl esters of liver microsomal lipids of CCl4-poisoned rats, was observed. As in the in vivo situation, the lipids recovered from this spot showed the absorption of conjugated dienes. On the other hand, when the incubation was carried out aerobically in the absence of EDTA either with CCl4 or without it, the g.l.c. analysis of the fatty acid methyl esters of microsomal lipids showed ECD responses which are probably due to the interaction of oxygen with unsaturated fatty acids. Ultraviolet spectra characteristic of peroxidized lipids were also found in this experimental condition. The activity of G-6-Pase, depressed by the aerobic incubation, could not be restored by the addition of different phospholipid fractions (phosphatidylcholine or phosphatidylethanolamine), denoting that during the incubation some irreversible damage to the enzyme activity occurs. The damage does not seem to be related to the alterations of the molecular structure of the membrane lipids (the binding of chlorinated radicals and the presence of conjugated dienes) since these alterations are also present after the anaerobic incubation, which does not cause a decrease in the G-6-Pase activity. Since the only experimental condition which produces an extensive loss of the enzyme activity is aerobic incubation, in which a large MDA formation occurs, it is concluded that some product evolved during the peroxidative breakdown of unsaturated lipids is responsible for the G-6-Pase inactivation.

The dialysate obtained from a system containing actively peroxidizing liver microsomes shows inhibitory effects on glucose 6-phosphatase activity of freshly prepared liver microsomes (test system). The inhibitory factors were recovered in extracts obtained from the dialysate with ethyl ether or ethyl acetate. The extraction procedure completely removed the inhibitory activity from the dialysate. A partial separation of the products present in the dialysate extract was obtained by thin-layer chromatography. When the lipid materials eluted from individual bands or groups of bands were tested for inhibitory activity, it was found that various Chromatographic bands contained various degrees of inhibitory activity, and that the highest inhibitory activity occurs in a well resolved band that is stained yellow by a N , N '-dimethyl- p -phenylene-diamine reagent. Additional studies indicated that this band contains most of the carbonyl functional groups detectable in the unfractionated dialysate extract, while it contains peroxide functional groups in trace amounts only. The peroxide functional groups present in the unfractionated dialysate extract were found to occur in various Chromatographic bands without a well defined relationship with the toxicological activity. It is concluded that lipoperoxidation products highly active in inhibiting microsomal glucose 6-phosphatase activity are fatty aldehydes or other carbonyl compounds, probably provided with a relatively long carbon chain.

Alterations produced by carbon tetrachloride in the lipids of extrahepatic tissues were studied. Consistent incorporation (about one-half of that occurring in the liver) of 14C from 14CCl4 was found in the phospholipids of the intestinal mucosa; kidney, adrenal and lung phospholipids were labeled to a lower extent, while spleen, testis, brain, heart and skeletal muscle lipids showed minor levels of radioactivity. The analysis of fatty acid methyl esters derived from phospholipids by gas-liquid chromatography (GLC) with an electron capture detector (ECD), showed ECD response similar to that observed with the liver, in the case of the intestinal mucosa and the kidney; on the contary no ECD response was seen with the organs, such as skeletal muscle, heart and testis in which the incorporation is almost negligible. Unlike liver phospholipids, the phospholipids of the intestinal mucosa and the kidney did not show diene conjugation absorption. Also, no evidence of lipid peroxidation was found in the thin-layer chromatographic analysis of fatty acid methyl esters prepared from the phospholipids of these organs. Possible meanings of these results are discussed.

When carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely 4-hydroxynonenal) are incubated with liver microsomes, a substantial amount of these products can be recovered bound to the microsomal protein, by allowing the microsomal protein to react with 2,4-dinitrophenyl-hydrazine. Concomitantly with the binding a decrease in the "exposed" and total -SH groups is stechiometrically accounted for by the amount of carbonyl compounds bound to the microsomal protein. Still in concomitance with the binding, the G-6-Pase activity of the microsomes markedly decreased. Therefore a mechanism similar to that described (12) for the binding of 4-hydroxyalkenals to -SH groups of protein and enzymes can be postulated.

In a study of the hepatic microsomal drug metabolising system in rats, pretreatment with cis-2-hydroxy-2-phenyl-cyclohexanecarboxilic acid (cicloxilic acid) did not prolong pentobarbital sleeping time or decrease the cytochrome P 450 content of the liver microsomes. The drug interacted with the liver microsomes to yield a type II spectral change. Cicloxilic acid did not affect the metabolism of CCl4 in vivo, as judged by the covalent binding of 14C from 14CCl r to microsomal lipids, or delay the gastrointestinal absorption of CCl4, as judged by the concentration of free CCl4 in the liver. The protection afforded by cicloxilic acid against CCl4 liver damage is therefore due neither to inhibition of the metabolism nor to delayed absorption of the toxin.

As a contribution to the understanding of the mechanisms by which addition of erythrocytes to liver microsomes incubated in the NADPH dependent system results in hemolysis, experiments are presented here in which the system containing actively peroxidizing microsomes was separated from the system containing the red blood cells by a dialysis membrane. It is shown that when liver microsomes were incubated with NADPH, lipid peroxidation, as measured by the formation of malonic dialdehyde (MDA), rapidly took place. The MDA concentration tended to equilibrate across the dialysis membrane. After a lag phase of 60 min, lysis of erythrocytes contained in the dialysis tube, started. It reached its maximal level (more than 80%) at 110 min of incubation. These results strongly suggest the possibility that toxic products originating during the course of lipid peroxidation induce pathological effect at distant loci. Furthermore, the accomplishment of a compartmentalization of the two systems involved in the reaction, may offer an approach to the recognition of the toxic factors.