James P. Kehrer - US grants

Pulmonary tissue which has received an acute toxic insult may either repair itself normally or develop a chronic fibrotic lesion characterized by the deposition of excess collagen. The inhibition of repair after the induction of acute lung damage enhances the development of fibrosis. Corticosteroids, which are the most widely used drug to treat patients with damaged lung tissue, have been reported to inhibit lung repair. This project will investigate the potential for fibrosis to develop in animals treated with corticosteroids after the induction of lung damage with butylated hydroxytoluene (BHT). Further studies will examine the effect of corticosteroids on collagen synthesis and degradation in damaged lung tissue. The rate at which collagen is synthesized in lung tissue in vitro is proportional to the extent of the initial damage. The relationship of these in vitro rates to the in vivo situation is not known. This project will compare rate of pulmonary collagen synthesis in vitro and in vivo. Collagen synthesis will be assessed by measuring the conversion of (3H) proline to (3H) hydroxyproline and by the specific digestion of labeled collagen by collagenase. These comparisons will be made in normal lung tissue and during the development of various levels of pulmonary fibrosis. Fibrosis will be generated by treating mice with BHT and subsequently exposing them to hyperoxia. Demonstrating that in vitro assays of the rate of lung collagen synthesis accurately reflect the in vivo situation will support the use of the more convenient in vitro assay to quantitate small amounts of lung damage. Additional experiments will assess the role of degradation of newly synthesized and established collagen in collagen homeostasis and pulmonary repair processes after acute lung damage and corticosteroid therapy. The degradation of newly synthesized collagen will be determined by measuring acid-soluble (3H) hydroxyproline following the administration of (3H) proline. The degradation of established collagen after various doses of BHT will be determined by measuring the rate at which (3H) hydroxyproline deposited following the intratracheal administration of (3H) proline is lost from lung tissue. The types of collagen synthesized at various times after the induction of lung damage and corticosteroid therapy will be determined by using an HPLC technique to separate collagen peptides following a cyanogen bromide digestion.

Pulmonary tissue which has received an acute toxic insult may either repair itself normally or develop a chronic fibrotic lesion characterized by the deposition of excess collagen. Treatment of patients with acute lung damage often involves the use of corticosteroids because of their anti-inflammatory activity and ability to decrease collagen synthesis in some in vitro and in vivo systems. Corticosteroid therapy of acute lung damage is controversial, however, because of reports it may be detrimental in some situations. The effects of corticosteroids in animal models of lung damage, as in humans, has been conflicting. It is not clear whether these differences are related to the species, type of lung damage, or steroid dosage regimen employed. Previous work has demonstrated that corticosteroids will enhance the development of fibrosis in mice with acute alveolar damage induced by butylated hydroxytoluene (BHT). This project will continue to investigate the effects of corticosteroids on collagen synthesis and degradation in this model system. However, BHT only damages lung tissue in mice. Therefore, potential differences in species responsiveness to corticosteroids will be studied using O,S,S-trimethyl phosphorodithioate (OSS), a chemical which produces a lung lesion much like BHT in rats, mice, and hamsters following intraperitoneal administration. Collagen synthesis in OSS- and BHT-damaged lung tissue will be assessed by measuring the conversion of (3H)proline to (3H)hydroxyproline and by the specific digestion of labeled collagen by collagenase. Additional experiments will determine the role of degradation of newly synthesized and established collagen in collagen homeostasis and pulmonary repair processes after the induction of acute damage and corticosteroid therapy. The degradation of newly synthesized collagen will be determined by measuring acid-soluble (3H)hydroxyproline after the administration of (3H)proline in vitro. The degradation of established collagen will be determined by measuring the rate at which (3H)hydroxyproline, deposited following the intratracheal administration of (3H)proline, is lost from lung tissue. The types of collagen synthesized at various times after the induction of lung damage and corticosteroid therapy will be determined by using a high performance liquid chromatographic technique to separate collagen peptides following cyanogen bromide digestion. The data obtained in mice, rats, and hamsters with OSS-induced lung damage will be compared to each other and to mice with BHT-induced lung damage.

Numerous cytotoxic drugs have been found to induce lung damage and pulmonary fibrosis. Although the underlying mechanisms are poorly understood, and may differ for each agent, the interaction of two antineoplastic drugs, or one such drug with previously damaged lung tissue, are two of several risk factors for the development of pulmonary fibrosis. The overall goals of this project are to determine the extent of lung damage resulting from the interaction of lung-toxic cytotoxic drugs with each other or with previously damaged lung tissue in mice, and to investigate some possible mechanisms for such interactions. Bleomycin and cyclophosphamide (CP) will be used since they have been used clinically in combination and each has been shown to produce lung damage in humans and animals. Studies will include 2 dose combinations of bleomycin and CP (including 2 doses of the same drug), or one dose in animals with existing lung damage. Preexisting lung damage, similar to that seen in some human lung lesions, will be induced in mice with O,S,S-trimethyl phosphorodithioate (OSS) and butylated hydroxytoluene (BHT). OSS is a systemic lung toxin that we recently showed produces a diffuse alveolar lesion in rats and mice similar to the BHT-induced lesion in mice. Two different "initiating" lung toxins will be used to investigate whether there are any toxin-specific interactions. Combination treatments may result in decreased, as well as increased, lung damage. Any treatments resulting in altered lung damage will be investigated to provide a better understanding of the mechanisms underlying each potential outcome of pulmonary interactions involving cytotoxic drugs. The extent of lung damage following various combination treatments will be assessed by measuring the total lung content of hydroxproline, an amino acid found primarily in collagen, and by histopathology. The time course of lung cell division, after single drug treatments, will also be used as an indirect measure of lung damage. These data will be considered when designing treatment schedules which involve a second agent since it has been hypothesized that pulmonary fibrosis develops when normal epithelial cell repair is inhibited or as the result of time-dependent additive lung damage. Mechanistic studies will involve correlating the effects of the non-lung-toxic and lung-toxic drug treatments with changes in pulmonary and hepatic mixed-function activities, pulmonary bleomycin hydrolase activity, pulmonary cell repair processes, and changes in the pulmonary glutathione system. Each of these factors has been implicated in modulating the pulmonary toxicity of CP or bleomycin and could be altered by preexisting lung damage or previous cytotoxic drug therapy thereby changing the pulmonary toxicity of subsequently administered doses of the same, or a different drug. Treatment regimens resulting in enhanced lung damage will also be used for studies on changes in collagen synthesis and the degradation of newly synthesized collagen. The results of these studies will reveal any differences in these parameters that are related to the species treated or the damaging agents employed.

An increased release of intracellular enzymes, indicative of cell death, has been observed upon reoxygenation of cardiac tissue following a sustained period of hypoxia (oxygen paradox). Two distinct pathways have been proposed to explain this phenomenon: a) the energy-dependent breakdown of cells injured during the preceding hypoxia, or b) the induction of additional damage by oxygen metabolites. Although evidence is available that oxygen radicals are formed upon reoxygenation and that antioxidants can protect against reperfusion injury, the presence of oxidative damage is not well established. The overall purpose of this project is to investigate some selected indices of oxidative stress in isolated-perfused heart tissue. Hearts from rats (which contain xanthine oxidase, a purported source of oxygen radicals) and from rabbits (which, like humans, lack this enzyme activity) will be used. Hearts will be perfused (Langendorff) for 30 min with oxygenated medium followed by 60 min of hypoxia and a further 30 min of oxygen. Cardiac oxygen uptake, lactate dehydrogenase release, left ventricular pressure and coronary flow rates will be monitored. The following indices of oxidative stress will be measured: a) the activity of sarcolemmal and sarcoplasmic reticulum (SR) calcium ATPase (this enzyme has been found to be susceptible to irreversible oxidative inhibition), b) alpha-tocopherol and alpha-tocopheryl quinone content, c) mixed disulfide and protein sulfhydryl content of whole heart, sarcolemmal and SR membranes, and d) cardiac conjugated diene and lipid hydroperoxide levels. Analyses will be performed in fresh heart, hearts perfused for 94 min with oxygenated medium, hearts perfused for 30 min with oxygenated medium followed by 60 min of hypoxia or 60 min of hypoxia plus 4 min of reoxygenation. Together, these studies will determine whether oxidative processes, which have been associated with cell death, are occurring in reoxygenated heart tissue. Preliminary data showed that cystamine, which oxidizes sulfhydryl groups and inhibits calcium ATPase in liver, can prevent myocyte lysis at reoxygenation. The effect of cystamine on sarcolemmal and SR protein sulfhydryls, mixed disulfides and calcium ATPase will be assessed. The role of calcium ATPase in controlling myocardial cell lysis at reoxygenation will be further studied by assessing the effect of 2 additional inhibitors of this enzyme, diamide and vanadium, on cardiac LDH release. Since inhibitors of ATP synthesis prevent enzyme release at reoxygenation, the effect of diamide, cystamine, and vanadate on myocardial energy content will also be measured. Finally, studies are proposed to investigate the ability of liposomally-entrapped ATP to mimic reoxygenation injury when infused 60 min of hypoxia and to prevent the oxygen paradox when infused throughout the hypoxic period. These studies will show whether the administration of exogenous ATP in a form which will enter cells is capable of protecting heart tissue from hypoxic injury or of stimulating enzyme release in hypoxic heart tissue not reoxygenated. Protection from hypoxic injury would have implications for improved organ preservation technology.

Cyclophosphamide (CP) is an orally active alkylating agent which must be bioactivated to exert its therapeutic and toxic effects. Although CP can be activated in vitro via mixed function oxidase (MFO-mediated 4- hydroxylation, a role for this system in the production of the active and toxic species in vivo has not been firmly established. The overall objectives of this proposal are to assess the hypothesis that alternative oxidation pathways, primarily cooxidation via prostaglandin H synthase (PHS), can also produce the 4-hydroxylated CP metabolite which is unstable and spontaneously breaks down to alkylating species, and to determine the importance of such metabolism in the lung and bladder toxicity of this drug. Preliminary studies have shown that inhibitors of PHS, but not MFO, activity prevents the development of lung damage in CP-treated mice. It was also found that arachidonic acid, as well as NADPH, could support the microsomal activation of CP to alkylating metabolite(s), and that CP could serve as a reducing cosubstrate in both lung and liver microsomes. Purified horseradish peroxidase, soybean lipoxygenase and ram seminal vesicle PHS, in the presence of arachidonic acid or H2O2, will be used as model systems to assess the ability of cooxidation reactions to metabolize CP. Analyses will include the loss of CP, the formation of water soluble species including acrolein, and covalent binding of 14C (ring) and 3H (chloroethyl side chain) labelled CP (which break down to labeled acrolein and phosphoramide mustard, respectively), to heat-inactivated microsomal protein. The metabolism and binding of CP by pure enzymes will be compared with metabolism and binding in tissues from ICR mice (sensitive to lung and bladder injury). Analyses will be done in vivo, in hepatic, kidney cortex and pulmonary microsomes, and in kidney papillae homogenates. In vivo studies will administer 200 mg/kg dual labelled CP ip. In vitro studies will initiate metabolism with NADPH, arachidonic acid or prostaglandin G2. Additional studies will assess the effect of specific cyclooxygenase, peroxidase, MFO and 5-lipogenase inhibitors on metabolism in vivo and on toxicity in vivo. Toxicity will be measured as changes in DNA synthesis and hydroxyproline content (lung) or blood content and gross morphology (bladder). The metabolism, cooxidation potential (using a synthetic hydroperoxide) and covalent binding of CP in tissues from ICR mice will be compared with that in C57 mice (resistant to lung and bladder injury) to obtain information on which factors are important for CP toxicity. Besides animal studies, experiments on the toxicity of CP to human lung cancer cell lines high or low in PHS activity will be done. CP is not directly toxic to cultured cells since it requires activation. The addition of arachidonic acid to cells high in PHS activity results in the activation of other agents. Since the same cell lines high in PHS also contain MFO activity, analyses will be performed with and without exogenous arachidonic acid and in the presence and absence of MFO and PHS inhibitors. The metabolism and covalent binding of CP in the presence and absence of these substances will be determined and correlated with cytotoxicity. Lastly, the effect of PHS inhibitors on the therapeutic activity of CP against tumor cell lines in mice will be assessed. The results of these studies will reveal the relative importance of cooxidation pathways for mediating the toxic and therapeutic effects of CP and could potentially lead to better targeting of tumors for CP therapy.

The ability of cells and tissues to defend against oxidative stress is determined by the supply of reducing equivalents. Our data demonstrate that cellular energy levels are not directly related to the ability of heart tissue to supply reducing equivalents in response to an oxidative stress. However, the pathways involved in this supply remain unclear, particularly if reactions which complete for reducing equivalents, such as ATP synthesis, are active. Studies in heart are of interest because of the sustained high respiration of this organ, and the potential for oxidative injury during reperfusion subsequent to infarction or surgery. Glutathione (GSH) is a major component of cellular antioxidant systems. GSH is maintained in the reduced form by glutathione reductase. Although this enzyme is specific for NADPH, the ability of intact cells, isolated mitochondria (which are a major source of free radicals and contain antioxidant systems independent of the rest of the cell), and whole tissues to supply reducing equivalents and maintain normal levels of GSH appears to involve NADH. The specific hypotheses to be tested are that: l) reducing equivalents used to reduce exogenous oxidizing agents can be supplied by NADH-linked substrates, 2) the supply of reducing equivalents to defend against oxidative stress is augmented under state 4 and diminished under state 3 respiration, and 3) intact organs can supply reducing equivalents in response to an oxidative stress more effectively than isolated cells. The validity of these hypotheses will be tested by using intact heart tissue, isolated cardiac mitochondria and cardiomyocytes provided with the following energy-linked substrates: glucose, acetate, lactate or pyruvate (intact heart and cardiomyocytes); malate/glutamate, succinate or octanoate (mitochondria). In each of these systems, the contents of ATP, phosphocreatine, NAD, NADH, NADP, and NADPH will be determined and correlated with levels of GSH, GSSH, protein thiols, and protein GSH-mixed disulfides. The effect of oxidatively stressing these systems with diamide (a direct thiol oxidant) or t-butyl hydroperoxide will be determined. The pathways by which reducing equivalents are supplied will be further assessed by providing the different energy-linked substrates in the presence of inhibitors of respiration (rotenone, antimycin A or FCCP [carbonyl cyanide p- trifluoromethoxyphenylhydrazone]). Mitochondrial substrate and inhibitor studies will be performed in both state 4 (without ADP) and state 3 (with ADP) respiration. These data will provide important new information on the pathways used by tissues to supply reducing equivalents and will reveal whether differences, which appear to exist between an intact organ and isolated cells, are real. The results may have important clinical relevance in the context of designing strategies for augmenting the ability of tissues to defend themselves against oxidative stress induced by reperfusion or xenobiotics.

Cyclophosphamide (CP) is a widely used alkylating agent which must be bioactivated to exert its therapeutic and toxic effects. There is a dose- limiting toxicity to the bladder and lung that may be caused by acrolein formed via a elimination reaction following an initial 4-hydroxylation of CP. Because metabolism occurs mainly in the liver, the mechanism by which this reactive aldehyde reaches the sites of injury is unknown. The overall hypothesis to be tested is that glutathione (GSH) conjugates or mercapturic acid metabolites of acrolein represent transport forms to deliver toxic CP metabolites to the sites of injury. Further processing may occur in the target organs prior to toxicity, or the conjugates themselves may be toxic. A secondary, but related, hypothesis is that nitrogen mustard metabolites of CP enhance the toxicity of these thiol metabolites in bladder. The specific aims to investigate these hypotheses are: 1) to assess the production of acrolein-glutathione metabolites in rats treated with CP. 2) to determine whether cells known to metabolize CP (hepatocytes), a tissue that is a target for CP toxicity (lung), and a lung cell line (A549) can generate toxic mercapturate(s) given the presumed precursor materials (acrolein or 3-oxopropylGSH). 3) to assess the general toxicity of 3-oxopropylGSH, 3-oxopropyl mercapturic acid and its S-oxide, and the di-acid forms [S-3-proprionic acid mercapturic acid 3-PrAMCA and its S-oxide] to these same systems as well as to isolated bladder tissue. 4) to determine the mechanism of toxicity of the various thiol metabolites. Whether it is the parent molecule, a breakdown product or a metabolite resulting from the oxidation of the aldehyde functionality to its acid will be determined. 5) to assess the mechanism of toxicity of CP as related to acrolein glutathione conjugates in vivo. Studies will be done in mice treated with CP or the glutathione metabolites it generates. Both lung and bladder toxicity will be evaluated with and without pretreatment with acivicin (gamma-glutamyl transpeptidase inhibitor), probenecid (organic anion transport inhibitor), hexahydrocoumarin (aldehyde oxidase inhibitor), and aminooxyacetic acid (Beta-lyase inhibitor). 6) to determine if and how nor-HN2 can enhance the bladder toxicity induced by these conjugates. The results of these studies may help devise new strategies to minimize the toxic effects of this drug.

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde to which humans are exposed in a variety of environmental situations. At low doses, acrolein inhibits cell proliferation without causing cell death and can enhance apoptosis from secondary toxins. The overall goals of this project are to determine the molecular mechanism(s) by which acrolein enhances apoptosis and inhibits proliferation. The hypothesis to be studied is that acrolein enhances apoptosis from secondary stressors including cell-cycle-non-specific anti-cancer drugs and that this is due to changes in genes or transcription factors regulating this process. Since acrolein is a metabolite of cyclophosphamide, this hypothesis leads to the conclusion that the unique effectiveness of this anti-cancer drug is a feature of both acrolein and the phosphoramide mustard metabolite. A related hypothesis is that the acrolein-mediated decrease in cell proliferation is caused by effecting changes in the expression of one or more growth- or stress-related genes or transcription factors secondary to a reduction in GSH which is rapidly depleted following acrolein treatment. The specific aims of this project to address these hypotheses are: 1) to assess in detail the ability of acrolein to enhance the susceptibility of cells to apoptosis form a secondary pro-apoptotic treatment; and 2) to determine the mechanism of the enhanced apoptosis and decreased proliferation following acrolein treatment. Since acrolein transiently depletes GSH, experiments will be run to differentiate effects directly related to a loss of antioxidant capacity to those related to changes in transcription factors or gene expression (even in those are secondary to the loss of GSH which may allow secondary agents better access to molecular targets). Preliminary data have implicated NF-kappaB as a target for acrolein. This will be further studied by determining the ability of diethyl maleate (DEM) (which blocks NF-kappaB activation at doses that do not deplete GSH) to affect apoptosis and by examining the effects of acrolein on NF-kappaB activation at doses that do not deplete GSH) to affect apoptosis and by examining the effects of acrolein on NF-kappaB and apoptosis in a second cell line. A role for other selected genes in the effects of acrolein will be determined. Specifically, the activation of AP-1 will be measured as well as any changes in mRNA levels and protein products of p53, c-jun, c-fos, c-myc, bcl-2, bcl-x, and bax at various times following acrolein insult. The results of these studies will provide important new information on pathways regulating cell proliferation and apoptosis, as well as provide data on the risk associated with low-level exposure to reactive species such as acrolein.

A relationship between reactive oxygen species and several effectors of apoptosis has been reported. Although the mechanism by which oxidants induce apoptosis is unknown, it is likely some signaling factor is generated. Since polyunsaturated fatty acids are highly susceptible to oxidation, oxidized products that are known to induce apoptosis are reasonable candidates. Lipoxygenase enzymes are a possible source of such products and various inhibitors have been used to implicate these enzymes in apoptosis. However, several of these inhibitors, including MK886 which blocks 5-lipoxygenase activating protein (FLAP), induce apoptosis even in cells lacking LOX. We have also shown that NM886 induces apoptosis independent of FLAP. Nevertheless, a link between FLAP and the BCL family of anti-apoptotic proteins is suggested by our findings that FL5.12 cells overexpressing bcl-xL have diminished levels of FLAP and there is a rapid loss of FLAP from such cells upon withdrawal of IL-3. There is also a rapid loss of bcl-xL and bcl-2 protein in cells treated with mK886. The overall goal of this project is to enhance our understanding of the mechanisms associated with apoptosis by determining the apoptotic mechanism of MK886. A related goal will be to clarify the relationship between FLAP and bcl-XL, and the induction of apoptosis. The hypotheses to be tested are that: 1) the inhibition of peroxisome proliferator activated receptors (PPAR) by MK886 plays a role in the induced apoptosis, 2) MK886 blocks the binding of unsaturated fatty acids increasing their cellular content. These fatty acids then activate apoptotic signaling pathways either directly or following conversion to other species, and 3) disrupting fatty acid signaling modulates the expression of bcl-xL. Preliminary data show that MK886 is a potent inhibitor of PPARalpha. This unique effect will be studied in more detail and any link to apoptosis investigated by up- and down-regulating PPAR and determining the apoptotic potency of MK886. The 2nd hypothesis will be studied by determining fatty acid oxidation after treatment with MK886, comparing the effects of MK886 to those of exogenous fatty acids including oxidized species, and examining whether glutathione can affect MK886-induced apoptosis. Any relationship between FLAP and bcl-XL will be determined by measuring the effect of bcl-XL overexpression on FLAP expression in other cell lines, assessing FLAP and bcl-XL protein and mRNA levels in bcl-XL overexpressing cells following withdrawal of IL-3 or treatment with MK886, and assessing the role of various proteolytic pathways in the loss of bcl-XL and FLAP. The results of these studies will improve our understanding of fatty acid signaling and apoptotic death in conjunction with FLAP and bcl proteins.

Description: The Pilot Projects Program is designed to meet three goals: 1) to support new investigators who show promise in becoming valuable assets to the Center; 2) to support established Center Investigators for exploratory projects of a highly innovative nature that are likely to attract new outside funding and; 3) to enhance interaction and collaboration of faculty both within and outside of the Center. The program is publicized by announcements to Center members, individual contact to nonmembers, and through departments throughout the campus. Applications are submitted once a year, reviewed by two scientists from outside the Center with ultimate funding decisions being made by the CRED Executive Committee. Applications are ranked based on scientific merit, relevance to Center goals, potential for extramural funding, and collaboration among Center members. Individual grants are generally $15,000 for 1 year up to $20,000. The Pilot Projects program will be modified in the future funding period by inviting applications that will target the following areas: animal model development, functional genomics, and molecular epidemiology.