Release of aldehydes from rat alveolar macrophages exposed in vitro to low concentrations of nitrogen dioxide.

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Inhibition of production of LTB4 and chemotactic agent from rat alveolar macrophages treated with t-butyl hydroperoxide is independent of ATP depletion.

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.

Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromatography mass spectrometry.

The detection of aldehydes has become an important measure of lipid oxidation in biological milieu. Aldehyde 2,4-dinitrophenylhydrazones are easily prepared and readily purified by HPLC and/or TLC and have proven useful for the detection of aldehydes. The lower limit of detection for dinitrophenylhydrazones was significantly reduced by using gas chromatography-mass spectrometric (GC-MS) techniques. Individual dinitrophenylhydrazones were readily separated by GC and detected by both positive and negative ion MS. The two major ions in negative ion spectra were the 182 m/z fragment ion and the molecular ion. Positive ion spectra showed strong ions corresponding to the protonated molecular ion and a protonated iminium ion. The greatest sensitivity was obtained with negative ion detection (10 pg per injection). However, more structural information was obtained from analysis of the positive ion spectra. Dinitrophenylhydrazones of hydroxyaldehydes, like 4-hydroxynonenal, were analyzed after converting the dinitrophenylhydrazones into trimethylsiloxylethers. GC-MS with negative ion detection was used to identify and quantitate the release of 4-hydroxynonenal by alveolar macrophages exposed to nitrogen dioxide.

Formation of tight monolayers of guinea pig airway epithelial cells cultured in an air-interface: bioelectric properties.

In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelectric properties of cultures grown in an air-interface were compared with those covered by medium. On day 1 for air-interface cultures, apical fluid was removed and basolateral fluid was replenished. For cultures covered by medium, varying volumes of apical fluid were used. On days 4 and 5 after plating, confluent GPTE monolayers in either liquid-covered or air-interface cultures exhibited similar monolayer resistance values > or = 1.0 kohm-cm2. However, the equivalent short-circuit current (Ieq) was significantly higher in air-interface cultures than those covered with medium on days 4 and 5. The Ieq in air-interface cultures on day 4 was 12.9 microA/cm2. These confluent GPTE cell monolayers cultured in air-interface could be a useful tool for studies of changes in bioelectric and ion transport properties in response to injury and mediators of inflammation.

Generation of glycolaldehyde from guinea pig airway epithelial monolayers exposed to nitrogen dioxide and its effects on sodium pump activity.

Pulmonary injury from nitrogen dioxide (NO2) may in part be related to the generation of aldehydic compounds, which bind with cellular proteins and subsequently impair or inhibit cell function. We examined the generation of aldehydes from guinea pig tracheobronchial epithelial (GPTE) cell monolayers exposed to NO2. With the use of dinitrophenylhydrazine (DNP) to derivatize aldehydic compounds, glycolaldehyde, a two carbon alpha-hydroxyaldehyde, was identified in elevated levels in the basolateral fluid from monolayers exposed to NO2. DNP-glycolaldehyde levels were 81.2 +/- 2.7 and 234.0 +/- 42.6 nM in response to a 1-hr exposure to 1 and 5 ppm NO2, respectively, as compared to an air-control value of 20.3 +/- 6.8 nM. Taking into account dilution and reactivity, cellular glycolaldehyde levels could have reached as high as 3 mM for the 60-min exposure period (i.e., 0.05 mM/min). The effects of exogenous glycolaldehyde on GPTE ouabain-sensitive basolateral 86Rb uptake (an index of Na+,K(+)-ATPase activity) were examined and compared with the actions of NO2 exposure. Bolus addition of glycolaldehyde to the basolateral fluid at concentrations > or = 5 mM led to an inhibition of ouabain-sensitive 86Rb uptake, while lower concentrations had no effect. the effects of exogenous glycolaldehyde differ from NO2 exposure, which led to a sustained elevation of ouabain-sensitive 86Rb uptake with presumed generation of glycolaldehyde at a continuous low level. Glycolaldehyde does not appear to play a significant role in the acute alterations of sodium pump activity, suggesting that the NO2-induced changes in Na+,K(+)-ATPase activity of GPTE monolayers probably are further mediated by other lipid peroxidation products/oxidation processes yet to be identified.

Effects of chronic administration of doxorubicin on plasma levels of prostaglandins, thromboxane B2, and fatty acids in rats.

The effects of multiple doses of doxorubicin (DXR) on hematocrit and plasma levels of prostaglandins (PG), thromboxane B2 (TxB2), total lipid, esterified and free fatty acids, and proteins were investigated in male rats. The rats received DXR (2 mg/kg) or vehicle weekly by the subcutaneous route for 2, 4, 8, and 13 weeks and were killed 1 week after their last dose. Another group of rats treated for 13 weeks was sacrificed at 19 weeks, 6 weeks after the last dose. No changes in hematocrit or plasma PG, TxB2, or total levels of fatty acids were noted between control and DXR-treated rats at either 3, 5, or 9 weeks. The hematocrit was slightly depressed from control levels at 14 and 19 weeks. Plasma PGE, PGF2 alpha, and TxB2 were elevated over control levels at 14 and 19 weeks. Plasma 6-keto-PGF1 alpha was increased over the control level only at 19 weeks. Total plasma lipid and esterified fatty acids were highly elevated over control levels at 14 and 19 weeks. Plasma free arachidonic acid was elevated over control levels at 14 and 19 weeks, while levels of other free fatty acids were unchanged. Plasma protein levels were slightly depressed from control levels at 3, 9, and 14 weeks. Elevations of plasma free arachidonic acid, PG, TxB2, esterified fatty acids, and total lipid might be involved in cardiotoxicity and nephrotoxicity found with chronic administration of DXR.

Effects of chronic administration of doxorubicin on myocardial beta-adrenergic receptors.

The effects of multiple doses of doxorubicin (DXR) on myocardial beta-adrenergic receptor density and dissociation constant were investigated in male Sprague Dawley rats. The rats received DXR (2 mg/kg) or vehicle weekly by the SC route for 13 weeks. One group of DXR-treated rats plus corresponding controls were sacrificed at 14 weeks, one week after the last dose. Another group of DXR-treated rats plus corresponding controls were sacrificed at 19 weeks, six weeks after the last dose. The myocardial beta-adrenergic receptor was characterized by radio-ligand binding studies using [125I]iodocyanopindolol. Beta--receptor densities in DXR-treated rats of 7.0 and 7.4 fm/mg protein were unchanged from control levels of 7.2 fm/mg protein at both 14 and 19 weeks, respectively. Receptor dissociation constants in DXR-treated rats of 36.7 and 36.9 pM were increased over control levels of 24.6 and 30.0 pM at 14 and 19 weeks, respectively. However, the change in dissociation constant is only significant at 14 weeks. The increased dissociation constants suggest diminished agonist binding affinity of the myocardial beta-receptor. This impaired response of the receptor to catecholamines would tend to diminish the ability of myocardium to adequately respond to adrenergic stimuli.

No change in alpha 1 adrenoceptors in canine femoral arteries after lumbar sympathectomy.

In the dog, alpha 1 adrenoceptors have been identified in the aorta, femoral mesenteric, and renal arteries. The concentration of alpha receptors may be regulated by hormones such as estrogen and progesterone and by drugs such as epinephrine. To assess whether sympathetic denervation, which is known to decrease norepinephrine output, might change the population of alpha 1 receptors in vascular smooth muscle, the femoral artery alpha 1 receptor population was examined 2 weeks after unilateral lumbar sympathectomy. [3H]Prazosin radioligand receptor assay analysis was used to measure the alpha 1 receptor populations. No statistical difference for dissociation constant and receptor density values between sympathectomy and nonsympathectomy femoral artery samples could be detected.

Dual effect of nitrogen dioxide on rat alveolar macrophage arachidonate metabolism.

Significant deficits in alveolar macrophage (AM) function have been associated with acute exposure to nitrogen dioxide (NO2). The present investigation examined changes in enzymatic production of arachidonate metabolites from rat AM exposed to NO2. While in vitro exposure of AM to NO2 concentrations between 0.1 and 5 ppm alone had small effects on basal synthesis of cyclooxygenase or lipoxygenase products, exposure to either 1 ppm (2 or 4 h) or 5 ppm (1 h) markedly enhanced the response of AM to stimulation by the calcium ionophore, A23187. This pre-exposure led to significant increases in cyclooxygenase products (thromboxane B2 (thromboxane), the stable metabolite of thromboxane A2, and 12-hydroxyheptadecatrienoic acid (12-HHT)) and lipoxygenase products (leukotriene B (LTB4) and monohydroxyeicosatetraenoate isomers) in response to A23187. In contrast, a 1-h exposure to 20 ppm NO2 alone significantly increased AM synthesis of thromboxane and 12-HHT, but suppressed the effect of subsequently added A23187. Increased synthesis of cyclooxygenase products with 20 ppm NO2 alone were blocked with the phospholipase inhibitor mepacrine and the cyclooxygenase inhibitor indomethacin. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) significantly reduced release of arachidonate; however, levels of thromboxane and 12-HHT were significantly increased. The results suggest a dual effect of NO2 on AM arachidonate metabolism in which low concentrations of NO2 had small effects on basal metabolism but markedly amplified the response to stimuli, while a high concentration of NO2 did the reverse. Such a complex dose-response effect may have significant impact in explaining the pathologic effects of NO2.

Dual effect of nitrogen dioxide on barrier properties of guinea pig tracheobronchial epithelial monolayers cultured in an air interface.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.

Air-interface cultures of guinea pig airway epithelial cells: effects of active sodium and chloride transport inhibitors on bioelectric properties.

To systematically study airway epithelial barrier properties and active ion transport processes during challenge to environmental toxicants, in vitro models that closely resemble airway epithelium in vivo are required. Guinea pig tracheobronchial epithelial (GPTE) cells cultured in an air interface form a tight, confluent monolayer, which may be a more suitable model for the studies of the airway epithelial barrier in vivo. In the present study, bioelectric properties of such GPTE cell monolayers were characterized with the use of pharmacological agents. Treatment of the basolateral side with ouabain completely abolished the short-circuit current (SCC), while apical addition had little effect. Apical addition of amiloride abolished 75% of the SCC, while basal addition had little effect. Treatment of the basolateral side with furosemide or bumetanide reduced the SCC by 20%, while apical addition had no effect. Apical or basolateral terbutaline increased SCC threefold with identical time courses; results with furosemide suggest a preferential stimulation of Cl- secretion. In contrast to GPTE monolayers, canine tracheal epithelial monolayers, under short-circuit conditions, primarily secrete Cl-, possibly due to apparent species differences. The predominance of Na+ reabsorption suggests that GPTE monolayers cultured in an air interface may share similar transport properties to human airway epithelium.

Effect of chronic administration of doxorubicin on cardiac adenylate cyclase activity in mice.

Cardiac adenylate cyclase activity was examined in mice treated chronically with doxorubicin. Mice received a subcutaneous dose of either 2 or 4 mg/kg doxorubicin twice weekly for 5 weeks. Mice were sacrificed five weeks after the last injection. Basal cardiac adenylate cyclase activity was significantly elevated in both the 2 and 4 mg/kg DXR-treated groups over the control level. GTP, isoproterenol (plus GTP), NaF, and forskolin stimulated activities in both the 2 and 4 mg/kg DXR-treated groups were also significantly elevated over control levels.

Enhancement of airway epithelial Na+,K(+)-ATPase activity by NO2 and protective role of nordihydroguaiaretic acid.

We examined the effects of nitrogen dioxide (NO2) on guinea pig tracheobronchial (GPTE) ouabain-sensitive 86Rb uptake, as an index of Na+,K(+)-ATPase activity, and specific [3H]ouabain binding. A 1-h exposure of GPTE monolayers to 5 ppm NO2 increased ouabain-sensitive 86Rb uptake (nmol.mg protein-1.30 min-1) to 512 +/- 39 compared with an air-control value of 278 +/- 20. Similarly, 1 ppm NO2 increased 86Rb uptake to 336 +/- 19 from an air control of 219 +/- 31. The specific [3H]ouabain-binding capacity (Bmax) for monolayers exposed to 5 ppm NO2 was increased to 23.2 +/- 1.2 pmol/mg protein in comparison with an air-control value of 18.4 +/- 0.4; however, there was no change at 1 ppm NO2. Binding constants (Kd) for 1 or 5 ppm NO2 were increased to 0.64 +/- 0.02 and 0.79 +/- 0.08 microM, respectively, in comparison with an air-control value of 0.53 +/- 0.02 microM. Changes of Bmax and Kd may be consistent with a recruitment of latent pumps to the basolateral cell plasma membrane and/or increased turnover of the sodium pump. However, the increase of Bmax was no more than 126% of the air control, while 86Rb uptake increased to 184%, suggesting that an increased turnover is the more predominant effect. Incubation of GPTE monolayers during NO2 exposure with nordihydroguaiaretic acid, an antioxidant, blocked the increase of ouabain-sensitive 86Rb uptake almost completely and partially protected transepithetial resistance, suggesting that lipid peroxidation processes may play a role in alterations of airway epithelial barrier and active ion transport properties.

Effects of chronic administration of doxorubicin on myocardial alpha-adrenergic receptors, histamine, cyclic nucleotides, calcium, norepinephrine, calmodulin, and guanylate cyclase activity, and plasma catecholamines in rats.

The present study examined changes in the levels of plasma catecholamines and myocardial histamine, guanylate cyclase activity, cyclic nucleotides, calcium, calmodulin, and norepinephrine following chronic administration of doxorubicin (DXR). In addition, changes in myocardial alpha 1-adrenergic receptor density and dissociation constant were measured. Rats received DXR (2 mg/kg) or vehicle weekly by the SC route for 2, 4, 8, and 13 weeks. Rats were sacrificed one week after their last dose. One group of rats treated for 13 weeks was sacrificed at 19 weeks, six weeks after the last dose. Heart histamine was unchanged at 3, 5, 9, and 19 weeks, yet at 14 weeks it was significantly elevated in DXR-treated rats over controls. Cardiac calcium, norepinephrine, and cyclic GMP levels were unchanged throughout the course of the study. Cardiac cAMP and calmodulin levels were unchanged at 3, 5, 9, and 14 weeks. At 19 weeks in DXR-treated rats, cAMP was depressed while calmodulin was elevated. Plasma catecholamines and myocardial guanylate cyclase activity examined at 14 weeks were unchanged. In contrast, alpha 1 receptor density examined at 14 weeks in DXR-treated rats was significantly depressed while the dissociation constant was unchanged. Changes in cAMP and calmodulin are suggestive of a redistribution of calcium, although total levels of calcium were unchanged. The depression of cAMP indicates damage to the membrane bound enzyme, adenylate cyclase, and that the membrane interaction of doxorubicin appears to be an integral part of the biochemical mechanism of its toxicity.

Effects of chronic administration of doxorubicin on heart phospholipase A2 activity and in vitro synthesis and degradation of prostaglandins in rats.

Male rats received doxorubicin (DXR) 2 mg/kg or phosphate buffered saline (PBS) weekly by the SC route for 13 weeks and were sacrificed at 14 and 19 weeks, one and six weeks, after the last dose, respectively. Heart phospholipase A2 activity in the 1000 X g supernatant was unchanged between DXR and PBS-treatment groups at both 14 and 19 weeks. In vitro heart microsomal syntheses of PGD2, PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were significantly elevated in DXR-treated rats over controls at 14 weeks. In contrast, syntheses of TxB2, PGE2, and PGF2 alpha in DXR-treated rats were significantly depressed from controls at 19 weeks. In vitro heart metabolism of PGF2 alpha in the 100,000 X g supernatant fraction was significantly elevated in DXR treated rats over controls at 14 weeks, but unchanged from controls at 19 weeks. It was concluded from the findings of the present study that increased prostaglandin synthesis may play a role in the mediation of cardiac injury induced by doxorubicin.

t-Butyl hydroperoxide stimulates alveolar macrophage biosynthesis of cyclooxygenase products.

Rat alveolar macrophages, labeled with 3H-arachidonic acid, were treated with t-butyl hydroperoxide (tBOOH). Treatment of cells with 100 microM tBOOH led to a rapid increase in 12-hydroxyheptadecatrienoic acid (12-HHT) within 2.5 minutes. At 15 minutes, 12-HHT levels appeared to plateau as there was no further increase at 30 minutes. TxB2 levels increased in a similar manner to that found with 12-HHT; however, only the level at 15 minutes was statistically increased. TxB2 levels also appeared to plateau at 15 minutes. Indomethacin, at a concentration of 1 microM, significantly inhibited TxB2 and 12-HHT production by approximately 90%. Desferal, an iron chelator, had no effect on alterations of biosynthesis of cyclooxygenase products by macrophages treated with tBOOH. No evidence of lipoxygenase products was found. Thus, these results suggest that tBOOH rapidly and selectively stimulated arachidonic acid metabolism through the cyclooxygenase pathway in rat alveolar macrophages. The stimulation of cyclooxygenase activity was transient with a maximum rate observed at 100 microM tBOOH.

Effect of ibuprofen on doxorubicin toxicity in mice.

This study examines the effect of ibuprofen on the toxicity produced in mice by the anticancer drug, doxorubicin. Ibuprofen solution made in 0.02% sodium carbonate, was administered subcutaneously in doses ranging from 10 to 40 mg/kg/day. Sodium carbonate served as a vehicle control and saline was used as a reference control. Treatment with saline, sodium carbonate, or a specific dose of ibuprofen began two days before administration of a single intraperitoneal dose of doxorubicin 17, 20 or 25 mg/kg and then continued for the next thirty days. Mortality was followed for thirty days. The analysis of the data by the Litchfield and Wilcoxon method revealed that there was neither a difference in doxorubicin LD50 values nor in the potency ratios between saline and any dose of ibuprofen treatment. Treatments with sodium carbonate and ibuprofen at 30 mg/kg/day offered slight protection over saline treatment, but it was not statistically significant. All mice, regardless of treatment, receiving doxorubicin showed significant weight loss. It appears that ibuprofen offers little protection against doxorubicin toxicity under these circumstances.

Modulation of ADP-stimulated inositol phosphate metabolism in rat alveolar macrophages by oxidative stress.

The present study examined alterations of adenosine-5-diphosphate (ADP)-stimulated inositol phosphate (IP3) metabolism and the respiratory burst of rat alveolar macrophages (AM) under oxidant stress using the model oxidant, tert-butyl hydroperoxide (tBOOH). Isolated AM were maintained in a system with an air-liquid interface, which approximates the lung environment. tBOOH produced a dual effect on the ADP-stimulated respiratory burst of these AM. Pretreatment of these AM with 50 microM tBOOH for 15 min led to a significant enhancement of the respiratory burst while significant inhibition was observed with 200 microM tBOOH. Treatment of AM with 100 microM ADP led to a significant increase in the generation of IP3, reaching a maximum at 30 s. In contrast, treatment of AM with 50 or 200 microM tBOOH did not stimulate IP3 generation during a 15-min period. Pretreatment of AM with 50 microM tBOOH for 15 min had no effect on ADP-stimulated IP3 generation. Preincubation with 200 microM tBOOH significantly inhibited generation of IP3 in response to ADP stimulation; however, this probably did not contribute to inhibition of the respiratory burst. The results suggest that production of IP3 may participate in stimulation of the respiratory burst by ADP but that enhancement of the respiratory burst by 50 microM tBOOH probably did not involve alteration of IP3 production.

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages.

The time course of uptake and distribution of 3H-arachidonic acid (3H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous 3H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateaus reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of 3H-AA into phosphatidylethanolamine was small, but continued to increase for 14 hours. Analysis of phosphate content in phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation of 3H-AA into PI. Cells were incubated with 3H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu Serum, or 0.1% BSA. Incubation of macrophages with 3H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of 3H-AA into phospholipids. Approximately 70% of incorporated 3H-AA was releasable through the action of exogenous phospholipase A2.