Use of mechanistic information to derive chemical-specific adjustment factors - Refinement of risk assessment.

When extrapolating data from animal toxicological studies a default factor (dUF) of 100 is applied to derive a heath based guidance value. The UF takes into account the interspecies differences (ID) and the intraspecies variability (IV). When re-evaluating the safety of phosphates used as food additives nephrocalcinosis was identified as the critical endpoint. The underlying mechanism for nephrocalcinosis was attributed to the precipitation of calcium phosphate in the kidney, depending on its solubility, irrespective of the species and the population. Based on the mechanism, the volume of primary urine, for which the glomerular filtration rate (GFR) was used as a proxy, was considered to be the only parameter relevant for ID and IV. Median value of GFR in rats was 4.0 ml/min/kg bw. In humans it was 1.6 ml/min/kg bw in healthy adults and 0.9 in elderly. These values were calculated from the distribution of the GFR data from 8 studies in rats (n = 191), 16 studies in adults (n = 1540) and 5 studies in elderly (n = 2608). Multiplying the distribution of the ratio rat/healthy humans (ID) with the distribution of the ratio healthy humans/elderly human (IV) resulted in a phosphate specific factor of 4.5 (3.3-6.7) (median; 25th - 75th percentile).

Mechanism of p-hydroxybenzoate ester-induced mitochondrial dysfunction and cytotoxicity in isolated rat hepatocytes.

The relationship between the metabolism and the cytotoxic effects of the alkyl esters of p-hydroxybenzoic acid (parabens) has been studied in freshly isolated rat hepatocytes. Incubation of hepatocytes with propyl-paraben (0.5 to 2.0 mM) elicited a concentration- and time-dependent cell death that was enhanced when enzymatic hydrolysis of propyl-paraben to p-hydroxybenzoic acid was inhibited by a carboxylesterase inhibitor, diazinon. The cytotoxicity was accompanied by losses of cellular ATP, total adenine nucleotide pools, and reduced glutathione, independently of lipid peroxidation and protein thiol oxidation. In the comparative toxic effects based on cell viability, ATP level, and rhodamine 123 retention, butyl- and isobutyl-parabens were more toxic than propyl- and isopropyl-parabens, and ethyl- and methyl-parabens and p-hydroxybenzoic acid were less toxic than propyl-paraben. The addition of propyl-paraben to isolated hepatic mitochondria reduced state 3 respiration with NAD+-linked substrates (pyruvate plus malate) and/or with an FAD-linked substrate (succinate plus rotenone), whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not affected significantly by propyl-paraben. Further, the addition of these parabens caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by propyl-paraben, butyl-paraben, and their chain isomers. These results indicate that a) propyl-paraben-induced cytotoxicity is mediated by the parent compound rather than by its metabolite p-hydroxybenzoic acid; b) the toxicity is associated with ATP depletion via impairment of mitochondrial function related to membrane potential and/or oxidative phosphorylation; and c) the toxic potency of parabens to hepatocytes or mitochondria depends on the relative elongation of alkyl side-chains esterified to the carboxyl group of p-hydroxybenzoic acid.

Hexamethylene diisocyanate (HDI)-induced lung impairment: studies in isolated perfused and ventilated guinea pig lungs.

Isolated, perfused and ventilated guinea pig lungs were exposed to hexamethylene diisocyanate via the air passages. Two air concentrations of hexamethylene diisocyanate were studied (3.5 and 11 mg/m3). There was a statistically significant (P < 0.05-0.001) dose-related reduction in both conductance and compliance but no effects were noted on the pulmonary circulation. With 3.5 mg/m3 hexamethylene diisocyanate the conductance capacity was reduced with 38% and compliance with 30% after 60 min. exposure. Eleven mg/m3 hexamethylene diisocyanate reduced the conductance and compliance capacity with 86 and 69%, respectively, on an average. The reduction in lung function (with 11 mg/m3) was abolished when 100 microM diclofenac, a cyclooxygenase inhibitor, was added to the perfusate (P < 0.01). The thromboxane A2 antagonist L-670, 596 (20 microM) exerted a partial protective effect. The capacity of conductance and compliance decreased with 46 and 32%, respectively, on an average, after preperfusion with L-670, 596 and a following exposure of 11 mg/m3 hexamethylene diisocyanate for 60 min. Statistically significant protection (P < 0.05) was obtained on compliance and the P-value was < 0.1 for conductance. Thus, these data indicate that hexamethylene diisocyanate-induced bronchoconstriction is mediated via arachidonic acid release and thromboxane formation, in isolated, perfused and ventilated guinea pig lungs.

Sodium metabisulfite and citric acid induce bronchoconstriction via a sulfite-sensitive pathway in the isolated guinea pig lung.

Inhalation of sodium metabisulfite (MBS; 80 mM; pH 2.9 +/- 0.1) or citric acid (CA; 0.4 M; pH 2.0 +/- 0.1) aerosols induced a reduction in compliance and conductance in the isolated perfused and ventilated guinea pig lung without affecting perfusion flow. The effect was dependent on the pH of the nebulized solution since inhalation of 80 mM MBS aerosols at pH 7.4 did not induce any effect on bronchial tone. Concomitantly to the bronchoconstriction induced by MBS or CA an increased level of calcitonin gene-related peptide (CGRP-LI) in the effluent perfusate was observed, indicating activation of sensory nerves. Sodium sulfite, a dissolution product of MBS, has previously been shown by our studies to reduce bronchoconstriction induced by inhalation of sulfur dioxide, in the isolated perfused and ventilated guinea pig lung. In the present study perfusion of the lung with sodium sulfite (3 mM) before and during exposure to aerosols with either MBS or CA attenuated the bronchoconstriction induced by the acidic solutions. The release of CGRP-LI induced by MBS or CA was not affected by sodium sulfite. Sulfite treatment did not modify perfused guinea pig lung reactivity towards acetylcholine (4 nmol), bradykinin (100 pmol), histamine (10 nmol), serotonin (500 pmol) and substance P fragment 5-11, a substance P analogue resistant to degrading enzyme (500 pmol). However, an inhibitory effect by sodium sulfite was observed on bronchoconstriction induced by the NK-2 agonist neurokinin A fragment 4-10 (NKA 4-10, 25 pmol). These results indicate that MBS- or CA-induced bronchoconstriction was dependent on the low pH of the aerosol solution and coincided with activation of sensory nerves. Sulfite modulation of the bronchoconstricting action of inhaled MBS and CA is suggested to be related to a sulfite-sensitive step in the signal transduction of the neuropeptide NKA.

Propyl gallate-induced DNA fragmentation in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with propyl gallate (PG) at concentrations of > or = 1 mM induced cell killing, whereas PG at < or = 0.5 mM did not cause cell death during a 3-h incubation. PG at > or = 0.5 mM elicited the ladder formation of soluble low-molecular weight DNA fragments with integer multiples of approximately 180 bp and specific nuclear DNA cleavages detected cytopathologically by labeling of a digoxigenin-nucleotide complex to new 3'-OH ends. Both of these PG-induced changes observed in hepatocytes are characteristic features of apoptosis. In contrast, the pretreatment of N-acetylcysteine (4 mM), a precursor of intracellular glutathione (GSH) and antioxidant, prevented PG (0.5 mM)-induced formation of soluble DNA fragments and loss of cellular GSH, ATP, and formation of blebbing. These results suggest that when the concentration of PG is decreased, the effects of PG on hepatocytes change from acute necrotic to apoptotic mode, and that the onset of DNA fragmentation is associated with GSH depletion.

Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes.

The relationship between cytotoxicity and mitochondrial dysfunction caused by propyl gallate (PG) has been studied in hepatocytes freshly prepared from fasted rats. Hepatocytes isolated from fasted (18 h) rats were significantly more susceptible to the toxicity of PG than hepatocytes from fed rats. The addition of fructose (15 mM), an alternative carbohydrate source, to hepatocyte suspensions resulted in the prevention of PG (1 mM)-induced cell killing accompanied by decrease in intracellular ATP loss during a 3 h-incubation period. Despite this, fructose did not completely prevent an abrupt loss of intracellular glutathione caused by PG, but effectively inhibited the loss of protein thiol levels. Fructose elicited a concentration (0.5-20mM)-dependent protection against the cytotoxicity of 1.5 mM PG. The incubation of hepatocytes with sodium azide (4 mM), an inhibitor of oxidative phosphorylation, enhanced the toxicity induced by PG (1 mM), but coincubation with fructose delayed the onset of toxicity. Neither azide alone nor fructose plus azide did affect the cell viability during the incubation period. Furthermore, the addition of 2 mM salicylamide, nontoxic to hepatocytes during the incubation period, enhanced PG (1 mM)-induced cytotoxicity and decreased the loss of free PG. These results indicate that the onset of cytotoxicity caused by PG may depend on the intracellular energy status and that mitochondria are critical target for the compound. In addition, the toxicity caused by the inhibition of mitochondrial ATP synthesis is related to the concentration of PG remaining in cell suspensions.

Age-related differences in the metabolism of sulphite to sulphate and in the identification of sulphur trioxide radical in human polymorphonuclear leukocytes.

Sulphite oxidation and sulphur trioxide radical formation were studied in polymorphonuclear leukocytes (PMNs) isolated from healthy young, old and centenarian donors and from patients with Down's syndrome. The sulphur radical formation measured by electron spin resonance spectroscopy-spin trapping (EPR-ST) was correlated with the activity of sulphite oxidase and with the rate of sulphite oxidation to sulphate by PMNs. Sulphite metabolism was studied both in resting, and phorbol myristate acetate (PMA) stimulated freshly isolated cells. The rate of sulphur trioxide radical formation was demonstrated by use of the spin trapping agent 5,5-dimethyl-1-pyroline-1-oxide (DMPO) with subsequent formation of an adduct. The intensity of adduct formation was most intense in cells with low sulphite oxidase activity, while a mixture of the adduct and of DMPO hydroxyl radical was mainly observed in cells with high sulphite oxidase activity. Furthermore, experiments carried out on purified sulphite oxidase showed that in the presence of sulphite the enzyme could also give rise to a DMPO-OH adduct. Sulphite oxidase activity in cells isolated from healthy young and old donors was positive correlated with both rates of sulphur trioxide radical formation and sulphite oxidation to sulphate, respectively. However, sulphite oxidase activity in cells isolated from centenarians and patients with Down's syndrome seems to loose partly its rate of oxidising sulphite to sulphate. The intensity of the sulphur centred radical adduct increased in the two latter groups of population and the radical observed was predominantly sulphur trioxide radical.

Peroxidase-catalyzed oxidation of 3,5-dimethyl acetaminophen causes cell death by selective protein thiol modification in isolated rat hepatocytes.

In this study we used a peroxidase model system (glucose/glucose oxidase and horseradish peroxidase) to investigate the effect of extracellularly generated reactive metabolites of 3,5-Me2-acetaminophen on cell viability and on cellular thiol levels. Incubation of hepatocytes with 3,5-Me2-acetaminophen in the presence of glucose/glucose oxidase and horseradish peroxidase caused a concentration-dependent loss of cell viability. Loss of viability was associated with decreased protein thiol levels. Addition of the reducing agent DTT, but not catalase, during the incubation restored cellular protein thiol levels and arrested the cell killing. Protein thiol depletion occurred selectively to the mitochondrial and microsomal fractions and was specific for a very limited number of protein bands. The data suggest that the oxidative modification of individual protein cysteine residues within the latter two organelle fractions is critically involved in the mechanism of toxicity.

Inverse relation between the concentration of low-density-lipoprotein vitamin E and severity of coronary artery disease.

Oxidation of low-density lipoprotein (LDL) is believed to play an important role in atherogenesis, and antioxidant vitamins are thought to protect against coronary artery disease (CAD). We investigated whether the vitamin E concentrations in serum and LDL were associated with the severity of CAD as assessed by a semiquantitative scoring system in which coronary angiograms are analyzed for the number and size of distinct stenotic lesions (global stenosis score). The study group consisted of 64 consecutive male survivors of myocardial infarction aged < 45 y. Lipid-adjusted serum and LDL vitamin E concentrations were significantly lower in the patients than in 35 age-matched male control subjects, whereas the absolute serum and LDL vitamin E concentrations did not differ significantly. No associations were found between the serum concentration or lipid-adjusted serum values of vitamin E and the stenosis score. In contrast, significant inverse correlation was found between the LDL vitamin E concentration, whether adjusted to the lipid (r=-0.477,P<0.001) or protein (r=-0.375, P<0.01) content of LDL, and the global coronary stenosis score. We conclude that a low LDL vitamin E concentration might play a role in the development of stenoses in coronary arteries and may contribute to clinically manifest CAD.

Effects of glutathione and pH on the oxidation of biomarkers of cellular oxidative stress.

Cellular oxidative stress is associated with such pathological conditions as arteriosclerosis, inflammatory diseases and cancer. The oxidation of the biomarkers. 2',7'-dichlorofluorescin (DCFH), 2-deoxyribose, and lipid peroxidation are often used to assess the status of oxidative stress in cells and tissues. Since high levels of reduced glutathione (GSH) and acidic conditions have been associated with diminished chemical lethality, we evaluated the influence of these parameters on the cellular response to oxidative stress. We used a cultured hepatocyte line (ch/ch cells) that is susceptible to oxidative toxicity. A hydroxyl radical-generating system consisting of H2O2, ascorbate and iron produced a pH-dependent lethality, with complete cell killing at pH 7.4 and none at pH 6.8. Lethality correlated with the depletion of intracellular GSH, and with an increase in DNA fragmentation. The influence of GSH and pH was assessed for DCFH and 2-deoxyribose oxidation, and for lipid peroxidation. The oxidation of DCFH and 2-deoxyribose was inhibited by GSH, with about 4-fold greater inhibition efficacy at pH 6.8 than at pH 7.4 [IC50 values (microM GSH) for pH 6.8 and 7.4, respectively: DCFH = 7 and 30; 2-deoxyribose = 125 and 490]. GSH did not affect lipid peroxidation at either pH, even at a high intracellular concentration of 10 mM. We conclude: 1) GSH is not inhibiting DCFH and 2-deoxyribose oxidation by simply quenching reactive oxygen (hydroxyl radical or perferryl oxygen), since GSH did not inhibit lipid peroxidation: 2) the protonated form GSH is more likely to be the inhibitory species rather than GS-, since even in the simple cell-free systems lower pH inhibited biomarker oxidation; and; 3) hydroxyl radical may not be the primary intracellular oxidant of DCFH, since intracellular GSH concentrations are typically 10- to 100-fold higher than the IC50 values for GSH inhibiting reactive oxygen-mediated DCFH oxidation.

Direct exposure to nitrogen dioxide fails to induce the expression of some inflammatory cytokines in an IC-21 murine macrophage cell model.

Biologically-active molecules secreted from alveolar macrophages, such as cytokines, have been proposed to be involved in the induction of pulmonary toxicity and inflammation in response to the inhalation of oxidant gas pollutants such as NO2 and O3. Despite this, mechanistic studies are hampered by the difficulty in obtaining control macrophages from human subjects, and the intrinsic variability of such primary cells. It is, thus, of importance to develop alternative models for such studies. Here, we have characterised expression kinetics of the mRNAs for tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), macrophage inflammatory protein-1 alpha (MIP-1 alpha) and macrophage inflammatory protein-1 beta (MIP-1 beta) in confluent cultures of the murine IC-21 macrophage line in response to LPS. The secretion of TNF-alpha protein into the medium, assayed by L-929 cell bioassay, closely followed the expression of its mRNA in response to the LPS stimulus. In contrast to LPS, the exposure of IC-21 cells to either air or various concentrations of NO2 in air between 2 and 20 ppm, in an inverted plate exposure model, failed to induce the expression of any of the cytokine mRNAs probed. We conclude that the IC-21 cell line may represent a suitable model for studying the role of stimulated cytokine gene expression in inflammation and that the early events in the pulmonary inflammatory response to the inhalation of NO2 do not involve stimulated release of TNF-alpha, IL-1 beta or MIP-1 alpha/MIP-1 beta from macrophages.

Analysis of the reactivity of [14C]toluene diisocyanate (TDI) in an isolated, perfused lung model.

An isolated, perfused, guinea pig lung model was used to investigate the molecular events which occur when a 14C-labeled TDI vapor reaches the airways. Exposure concentrations of 0.2 and 0.7 ppm were tested. Perfusate composition included: Krebs Ringer buffer only, as well as buffer containing either guinea pig serum albumin, human serum albumin, or diluted guinea pig plasma. Radioactivity was detected in the perfusate within minutes of exposure, and following a delay, increased linearly. The rate of uptake was dependent on TDI concentration and the composition of the perfusate. Biochemical characterization of the state of the 14C-labeled material in the perfusate was performed. The distribution between low and high molecular weight reaction products was determined by molecular sieve fractionation and varied as a function of perfusate composition but no variability was observed as a function of time during the 45 min of exposure. An increase in nucleophile concentration in the perfusate was associated with both a higher percentage of conjugated products (from 15% with buffer only to 45% with diluted guinea pig plasma) and an increase in the rate of TDX uptake (from 0.5 microns Eq/min with buffer alone to 0.1 micrograms Eq/min with diluted GPSA as perfusate at 0.7 ppm). GC-MS analysis of the samples for free TDA, before and after acid hydrolysis, showed that the low molecular weight product(s), which represented from 55-85% of the circulating radioactivity, was composed of hydrolyzable and non-hydrolyzable conjugates and metabolites with approximately 4% of the label associated with free TDA. Although the distribution between high and low molecular weight species varies, this result is analogous to the findings from in vivo studies and suggests that the isolated, perfused lung (IVPL) system may be a useful tool in investigating the molecular mechanisms of isocyanate-induced disease and metabolic activity of the lung.

Evidence for the activation of the signal-responsive phospholipase A2 by exogenous hydrogen peroxide.

The intracellular events that lead to arachidonic acid release from bovine endothelial cells in culture treated with hydrogen peroxide were characterized. The hydrogen peroxide-stimulated release of arachidonic acid was time- and dose-dependent, with maximal release achieved at 15 minutes after the addition of 100 microM hydrogen peroxide. Hydrogen peroxide-stimulated release of arachidonic acid was blocked with the phospholipase A2 inhibitor quinacrine. Treatment of the cells with hydrogen peroxide did not result in liberation of oleic acid, indicating that hydrogen peroxide exercised its effect on an arachidonate-specific phospholipase. Pretreatment of the cells with antioxidants, transition metal chelators, and hydroxyl radical scavengers did not affect the hydrogen peroxide-stimulated arachidonic acid release, indicating that the response to hydrogen peroxide is not oxygen radical-mediated. The response to hydrogen peroxide does not appear to be calcium-dependent, due to the following two observations: (a) No increase in intracellular calcium was seen upon exposure of the FURA2-loaded cells to hydrogen peroxide at concentrations sufficient to release arachidonic acid, and (b) no change in the release response was detected in cells loaded with the intracellular calcium chelator BAPTA. Significant inhibition of arachidonic acid release was seen when the cells were pretreated with inhibitors of protein kinase C, but not with inhibitors of tyrosine kinase. The results of these studies indicate that hydrogen peroxide-stimulated arachidonic acid release is mediated by a specific signal-responsive phospholipase A2, and that this process is not mediated via the actions of either lipid peroxidation or calcium but, rather, that a stimulation of intracellular kinase activity is necessary for this response.

Metabolism and cytotoxicity of propyl gallate in isolated rat hepatocytes: effects of a thiol reductant and an esterase inhibitor.

The relationship between the metabolism and the cytotoxic effects of propyl gallate (PG) has been studied in freshly isolated rat hepatocytes. Addition of PG (0.5-2.0 mM) to the hepatocytes elicited concentration-dependent cell death, accompanied by decreases in intracellular ATP, adenine nucleotide pools, glutathione, and protein thiols. The rapid loss of ATP preceded the onset of cell death. PG in the hepatocyte suspensions was converted to gallic acid, 4-O-methyl-gallic acid, and other minor products over time. In addition, PG was converted to a dimer [dipropyl-4,4',5,5',6,6'-hexahydroxydiphenate (PG-dimer)] and ellagic acid via autooxidation. In comparisons of the toxic effects of PG and its metabolites at concentrations of 2 mM, the parent compound PG was the most toxic. Pretreatment of hepatocytes with diazinon (100 microM), an esterase inhibitor, enhanced PG-induced cytotoxicity. This was accompanied by delay of PG loss and inhibition of gallic acid formation. The cytotoxicity of PG was also enhanced by addition of the thiol reductant dithiothreitol (4 mM), although intracellular levels of glutathione and protein thiols were maintained during the incubation period. Dithiothreitol did not affect the hydrolysis of PG to gallic acid by esterases but did delay the conversion of PG and prevented the formation of PG-dimer. In isolated hepatic mitochondria, PG elicited a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. In contrast, PG-dimer inhibited the rate of state 3 oxygen consumption. Based on the respiratory control index, the order of potency for impairment of mitochondria was PG > PG-dimer > gallic acid = 4-O-methyl-gallic acid = ellagic acid - propyl alcohol. These results indicate (a) that PG-induced hepatotoxicity is mediated by the parent compound and not its metabolites, (b) that toxicity is associated with ATP depletion apparently independently of cellular thiol depletion, and (c) that mitochondria may represent critical targets of PG-induced cytotoxicity.

The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells.

The depletion of human umbilical vein endothelial (HUVE) cell glutathione with buthionine sulfoximine or with sulfur amino acid-free medium potentiated the sub-lethal (3H-deoxyglucose release) and lethal (lactate dehydrogenase release) cytotoxicity responses of the cells to direct exposure to NO2 over the range 2-20 ppm. When control cells, or glutathione-depleted cells, were either pre-loaded with ascorbate (intracellular ascorbate), or washed with ascorbate-containing medium just before exposure (extracellular ascorbate), the cells were fully protected from NO2-dependent toxicity. Concomitant with these exposures, NO2 caused dose-dependent depletions of both glutathione and ascorbate. Further, it was noted that the depletion of the intracellular ascorbate pool was accelerated in these glutathione depleted cells. Conversely, loading ascorbate into the cells significantly diminished NO2-dependent depletion of intracellular GSH. In contrast to affecting the acute cytotoxicity response of the HUVE cells to NO2, ascorbate supplementation of the medium of cells exposed to NO2 at clonal density facilitated considerable protection to the colony-forming efficiency of the cells. We conclude that both ascorbate and glutathione play important protective roles in defending HUVE cells from the toxicity of NO2 under direct exposure conditions. The results also strengthen the premise that ascorbate and glutathione co-operate in the antioxidative protection of cellular viability.

Characterization of bronchodilator effects and fate of S-nitrosothiols in the isolated perfused and ventilated guinea pig lung.

In this study the effects of S-nitrosothiols, in particular S-nitrosoglutathione (GSNO), were evaluated with regard to their bronchodilating properties, both after infusion via the pulmonary circulation and after inhalation, in the isolated perfused and ventilated guinea pig lung. Infused GSNO induced bronchorelaxation of lungs that were precontracted with methacholine. During a 15-min period of single-passage perfusion with GSNO (10 microM), maximally 10% was taken up and/or degraded by the lung. A spontaneous breakdown of GSNO in the perfusion buffer was also observed, which was partially accompanied by the formation of nitrite. Low levels of nitric oxide (NO) were detected in the perfusion buffer when GSNO was present. This was due to the presence of contaminating transition metals, because EDTA and 2,2'-dipyridyl largely reduced the formation of NO. The NO-scavenging agents oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide abolished levels of NO in the buffer but did not abolish GSNO-induced bronchodilation. The effects of infused GSNO are therefore attributed to an action of the intact S-nitrosothiol and not to NO released from GSNO in the perfusion buffer. Similarly, perfusion with S-nitrosated glutathione isopropyl ester, cysteinyl glycine, N-acetyl-L-cysteine or N-acetyl-D,L-penicillamine, but not with nitrosated bovine serum albumin or sodium nitrite, was found to induce bronchodilation. Inhalation of nebulized GSNO induced bronchodilation of methacholine-precontracted lungs with a rapid onset of action, although it was a less potent bronchodilator than salbutamol. The results show that infused or inhaled S-nitrosothiols have bronchodilating properties in the isolated perfused and ventilated guinea pig lung.

Studies on the effect of sulfite on benzo[a]pyrene-7,8-dihydrodiol activation to reactive intermediates in human polymorphonuclear leukocytes.

Sodium sulfite, a hydrolysis product of the environmental pollutant sulfur dioxide increased the activation of (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) to the (+)-anti-enantiomer of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) in phorbol myristate acetate (PMA)-stimulated human polymorphonuclear leukocytes (PMNs). This effect was potentiated in the presence of DMSO. No significant effect of sulfite on BP-7,8-diol activation was observed in resting leukocytes. As revealed by the 32P-postlabelling technique the dominant adduct in both intracellular DNA and to DNA added to the leukocytes was (+)-anti-BPDE bound to the exocyclic nitrogen of deoxyguanosine. The mechanism underlying the stimulatory effect of sulfite on diol epoxide production and increased DNA-binding probably involves one-electron oxidation of sulfite to a sulfur trioxide radical anion and subsequent reaction with molecular oxygen to form the corresponding peroxyl radical. This step obviously requires PMA-initiated oxidative burst and thus, production of superoxide radical anions (O2-.).

Cytotoxicity of NO2 gas to cultured human and murine cells in an inverted monolayer exposure system.

We report the development of an optimised exposure system for the exposure of inverted cell cultures to NO2, which presents several advantages over conventional, right-side-up exposure systems. Firstly, the cells may be directly exposed to NO2 in the gas phase for up to 1 h, without the interposition of an aqueous layer. Secondly, the chamber system allows simple and precise control of the gas concentration during the exposure. Finally, the system allows the simultaneous exposure of large numbers of cells under sterile conditions, facilitating further culture of the cells after the exposure period. We report the application of this system to a comparative study of the toxicity of NO2 in three different cell types involved in the circuit of the inflammatory response, the IC-21 murine macrophage line, the A-549 human pulmonary type II-like epithelial cell line and human umbilical vein endothelial cells. As little as 2 ppm NO2 for 20 min reduced colony-forming efficiency of HUVE cells and A-549 cells and A-549 cells to 35% and 78% of their air controls, respectively. Exposure to 5 ppm NO2 for 1 h increased lactate dehydrogenase release of HUVE cells, IC-21 macrophages and A-549 cells from 7.9% to 21.6%, 5.7% to 10.9% and 2.0% to 3.4%, respectively, whilst 10 ppm NO2 for 1 h lowered cellular glutathione in HUVE cells, IC-21 cells and A-549 cells from 35.2 nmol/mg to 23.3 nmol/mg, from 45.0 nmol/mg to 31.0 nmol/mg and from 86.4 nmol/mg to 69.2 nmol/mg, respectively. Of the cell types tested it was shown that HUVE cells and IC-21 cells were equally sensitive to the toxicity of NO2, whilst A-549 cells displayed considerable resistance, perhaps due to the considerably higher levels of glutathione in this cell line. Further, a comparison of the sensitivity of HUVE cells to NO2, using several modes of exposure (inverted and right-side-up (either rocked or static)) and the assay of lactate dehydrogenase and [3H]deoxyglucose release, revealed that the present inverted exposure technique potentiated the acute cytotoxicity of the gas.

N-ethyl maleimide stimulates arachidonic acid release through activation of the signal-responsive phospholipase A2 in endothelial cells.

Treatment of bovine endothelial cells with the alkylator N-ethyl maleimide results in arachidonic acid mobilization. N-ethyl maleimide-stimulated arachidonic acid release was dose and time dependent and maximum release was achieved after 10-15 min with 50 microM N-ethyl maleimide, N-ethyl maleimide-stimulated arachidonic acid release could be prevented by pretreating the cells with the phospholipase A2 inhibitor quinacrine. Based on the finding that N-ethyl maleimide was not able to release oleic acid from oleic acid-preloaded cells, it was clear that the effect of N-ethyl maleimide was limited to an arachidonic acid-specific phospholipase. The effect of N-ethyl maleimide does not appear to be dependent on calcium, as shown by the observation that N-ethyl maleimide was not able to increase intracellular calcium concentration in FURA2-loaded cells. Pretreatment of the cells with staurosporine totally inhibited N-ethyl maleimide-stimulated arachidonic acid liberation. The tyrosine kinase inhibitor genistein was also able to significantly inhibit arachidonic acid release. It is concluded from the results obtained in this study that N-ethyl maleimide stimulates arachidonic acid release by stimulating the activity of a specific, signal-responsive phospholipase A2. Furthermore this activation is not mediated by intracellular calcium fluxes but by a stimulation of intracellular kinase activity which eventually leads to the activation of this signal-responsive phospholipase A2.