Restoration of sensitivity to oxazaphosphorines by inhibitors of aldehyde dehydrogenase activity in cultured oxazaphosphorine-resistant L1210 and cross-linking agent-resistant P388 cell lines.

The sensitivity of cultured L1210 and P388 cells sensitive (L1210/0, P388/0) and resistant (L1210/OAP, P388/CLA) to oxazaphosphorines, to 4-hydroperoxycyclophosphamide, ASTA Z-7557, phosphoramide mustard, and acrolein was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. The L1210/OAP cell line is resistant specifically to the oxazaphosphorines; P388/CLA cells are partially cross-resistant to other cross-linking agents. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines, 4-hydroperoxycyclophosphamide and ASTA Z-7557, against L1210/OAP and P388/CLA cells; in the presence of a sufficient amount of inhibitor, sensitivity was essentially fully restored in both cases. The inhibitors did not potentiate the cytotoxic action of the nonoxazaphosphorines, phosphoramide mustard and acrolein, against these cell lines. The cytotoxic action of the oxazaphosphorines and nonoxazaphosphorines against L1210/0 and P388/0 cells was not potentiated by any of the aldehyde dehydrogenase inhibitors. Inhibitors of xanthine oxidase or aldehyde oxidase activities did not potentiate the cytotoxic action of the oxazaphosphorines against L1210/OAP cells. These observations strongly suggest that (a) aldehyde dehydrogenase activity is an important determinant with regard to the sensitivity of a cell population to the oxazaphosphorines; (b) L1210/0 and P388/0 cells lack the relevant aldehyde dehydrogenase activity; (c) the phenotypic basis for the resistance to oxazaphosphorines by L1210/OAP cells is aldehyde dehydrogenase activity; and (d) the major reason that P388/CLA cells are resistant to oxazaphosphorines is aldehyde dehydrogenase activity.

Potentiation of the cytotoxic action of mafosfamide by N-isopropyl-p-formylbenzamide, a metabolite of procarbazine.

Several mouse aldehyde dehydrogenases catalyze the detoxification of aldophosphamide, the pivotal metabolite of the prodrugs cyclophosphamide, mafosfamide, and other oxazaphosphorines. N-Isopropyl-p-formylbenzamide, a major metabolite of procarbazine, was found to be an excellent substrate (Km = 0.84 microM) for at least one of these enzymes, namely, mouse aldehyde dehydrogenase-2. The Km for mouse aldehyde dehydrogenase-2-catalyzed detoxification of aldophosphamide is 16 microM. Thus, competition between N-isopropyl-p-formylbenzamide and aldophosphamide for the catalytic site on the enzyme should strongly favor the former, and the rate at which aldophosphamide is detoxified should be markedly retarded. Mouse L1210/OAP and P388/CLA leukemia cells are relatively insensitive to the oxazaphosphorines because they contain large amounts of mouse aldehyde dehydrogenase-2. As predicted, N-isopropyl-p-formylbenzamide markedly potentiated the cytotoxic action of mafosfamide against these cells. Mouse L1210/0 and P388/0 lack the enzyme. Again as expected, N-isopropyl-p-formylbenzamide essentially did not potentiate the cytotoxic action of mafosfamide against these cells. Certain mouse and human hematopoietic progenitor cells also contain an aldehyde dehydrogenase that catalyzes the detoxification of aldophosphamide, but the specific identity of this enzyme remains to be established. N-Isopropyl-p-formylbenzamide potentiated the cytotoxic action of mafosfamide against these cells as well. Clinically, procarbazine and the oxazaphosphorines are used to treat certain neoplastic diseases. Frequently, they are used in combination. Our findings demonstrate the potential for both desirable and undesirable drug interactions when these agents are used concurrently. Similar drug interactions can be expected when other substrates for, or inhibitors of, the relevant aldehyde dehydrogenases, e.g., chloramphenicol, chloral hydrate, and methyltetrazolethiol-containing cephalosporins, are co-administered with the oxazaphosphorines.

Identification of a methylcholanthrene-induced aldehyde dehydrogenase in a human breast adenocarcinoma cell line exhibiting oxazaphosphorine-specific acquired resistance.

The class-3 aldehyde dehydrogenase that is overexpressed (> 100-fold) in human breast adenocarcinoma MCF-7/0 cells made resistant (> 30-fold as judged by LC90s) to oxazaphosphorines, such as mafosfamide, by growing them in the presence of polycyclic aromatic hydrocarbons, e.g., methylcholanthrene (3 microM for 5 days), was isolated and characterized. Its physical and catalytic properties were identical to those of the prototypical human stomach mucosa cytosolic class-3 aldehyde dehydrogenase, type-1 ALDH-3, except that it catalyzed, though not very rapidly, the oxidation of aldophosphamide, whereas the stomach mucosa enzyme essentially did not; hence, it was judged to be a slight variant of the prototypical enzyme. Carcinogens that are not ligands for the Ah receptor, barbiturates known to induce hepatic cytochrome P450s, steroid hormones, an antiestrogen, and oxazaphosphorines did not induce the enzyme or the largely oxazaphosphorine-specific acquired resistance. Whereas methylcholanthrene induced (a) resistance to mafosfamide and (b) class-3 aldehyde dehydrogenase activity, as well as glutathione S-transferase and DT-diaphorase activities, in the estrogen receptor-positive MCF-7/0 cells, it did not do so in two other human breast adenocarcinoma cell lines, MDA-MB-231 and SK-BR-3, each of which is estrogen receptor negative. Expression of the class-3 aldehyde dehydrogenase and the loss of sensitivity to mafosfamide by polycyclic aromatic hydrocarbon-treated MCF-7/0 cells were transient; each returned to essentially basal levels within 15 days when the polycyclic aromatic hydrocarbon was removed from the culture medium. Insensitivity to the oxazaphosphorines on the part of polycyclic aromatic hydrocarbon-treated MCF-7/0 cells was not observed when exposure to mafosfamide (30 min) was in the presence of benzaldehyde or octanal, each a relatively good substrate for cytosolic class-3 aldehyde dehydrogenases, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for these enzymes. These observations demonstrate that ligands for the Ah receptor can induce a transient, largely oxazaphosphorine-specific, acquired cellular resistance, and they are consistent with the notion that elevated levels of a cytosolic class-3 aldehyde dehydrogenase nearly identical to the prototypical type-1 class-3 aldehyde dehydrogenase expressed by human stomach mucosa account for the Ah receptor ligand-induced oxazaphosphorine-specific acquired resistance, most probably by catalyzing the detoxification of aldophosphamide.

Collateral sensitivity to cross-linking agents exhibited by cultured L1210 cells resistant to oxazaphosphorines.

The sensitivity of cultured L1210 and P388 cells, sensitive (L1210/0, P388/0) and resistant (L1210/CPA, P388/CPA) to cyclophosphamide in vivo, to five oxazaphosphorine and eight nonoxazaphosphorine cross-linking agents was determined. Each of the resistant sublines was cross-resistant to all of the oxazaphosphorines tested. The P388/CPA cell line was also cross-resistant to all of the nonoxazaphosphorines but, in most cases, not nearly to the same extent. The L1210/CPA cell line was collaterally sensitive to all but one of the nonoxazaphosphorines, in which case it was equisensitive. Changes in sensitivity could not be accounted for by changes in intracellular pH values, or by changes in intracellular inorganic phosphate or acid-soluble organic phosphate concentrations. Inasmuch as the L1210/CPA cell line was specifically resistant to the oxazaphosphorines, identification of the phenotypic basis for this resistance should serve to identify a potentially important determinant with regard to the basis for the oncotoxic specificity of this group of agents.

Identification of the mouse aldehyde dehydrogenases important in aldophosphamide detoxification.

Aldophosphamide, the penultimate cytotoxic metabolite of cyclophosphamide, can be detoxified by an oxidation reaction catalyzed by certain aldehyde dehydrogenases. The selective toxicity of cyclophosphamide is due, at least in part, to a greater expression of the relevant aldehyde dehydrogenase activity in normal cells relative to that expressed in certain tumor cells. Not known at the onset of this investigation was which of the several known mouse aldehyde dehydrogenases catalyze this reaction. Twelve enzymes that catalyze the NAD(P)-linked oxidation of aldophosphamide, acetaldehyde, benzaldehyde, and/or octanal were chromatographically resolved from mouse liver. Four of these appear to be novel; four others were determined to be betaine aldehyde dehydrogenase, succinic semialdehyde dehydrogenase, glutamic gamma-semialdehyde dehydrogenase, and xanthine oxidase (dehydrogenase). An additional aldehyde dehydrogenase, namely AHD-4, was semipurified from stomach. The stomach enzyme and nine of the hepatic enzymes catalyze the oxidation of aldophosphamide. Km values for these reactions range from 16 microM to 2.5 mM. The relevant aldehyde dehydrogenase of major importance varies with the tissue. In the liver, the major cytosolic aldehyde dehydrogenase, namely AHD-2, accounts for greater than 60% of total hepatic aldehyde dehydrogenase-catalyzed aldophosphamide (160 microM) detoxification. Succinic semialdehyde dehydrogenase (AHD-12) and three of the novel hepatic aldehyde dehydrogenases, namely AHD-8, AHD-10, and AHD-13, also contribute significantly to total hepatic aldehyde dehydrogenase-catalyzed aldophosphamide detoxification. In the stomach, AHD-4 and AHD-8 account for approximately 86% of total aldehyde dehydrogenase-catalyzed aldophosphamide (160 microM) detoxification. AHD-2 was not found in this tissue. Of all the aldehyde dehydrogenases examined, AHD-2 and AHD-8 were estimated to be the most efficient catalysts of aldophosphamide oxidation. Thus, these enzymes would seem most likely to be operative when tumor cells acquire aldehyde dehydrogenase-mediated cyclophosphamide resistance.

Conversion of 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein mediated by bifunctional catalysis.

The rates at which 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer composition, buffer concentration, and pH. Conversion of 4-hydroperoxycyclophosphamide to 4-hydroxycyclophosphamide in 0.5 M Tris buffer, pH 7.4, 37 degrees, was first-order (k = 0.016 min-1), but subsequent conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein under these conditions was negligible. Phosphoramide mustard and acrolein were readily generated from 4-hydroperoxycyclophosphamide or 4-hydroxycyclophosphamide when either of these agents was placed in phosphate buffer. Conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was first-order with respect to 4-hydroxycyclophosphamide (k = 0.126 min-1 in 0.5 M phosphate buffer, pH 8, 37 degrees) as well as first-order with respect to phosphate serving as a catalyst. The rate-determining step in the reaction was pH dependent only insofar as the hydrogen ion concentration governed the relative amounts of monobasic and dibasic phosphate present. Pseudo-first-order rate constants were 0.045 M-1 min-1 for monobasic phosphate and 0.256 M-1 min-1 for dibasic phosphate. The role of phosphate in this reaction was as that of a bifunctional catalyst. The reaction was not subject to specific or general, acid or base, catalysis. Other bifunctional catalysts such as glucose-6-phosphate and bicarbonate also catalyzed the reaction, albeit less efficiently. Aldophosphamide apparently exists only transiently; its presence could not be established by 31P nuclear magnetic resonance spectroscopy. We conclude that, in the reaction sequence 4-hydroxycyclophosphamide leads to aldophosphamide leads to phosphoramide mustard + acrolein, the conversion of 4-hydroxycyclophosphamide to aldophosphamide is rate limiting and is subject to bifunctional catalysis; this reaction can proceed efficiently only in the presence of a bifunctional catalyst. Assuming that the oncotoxic specificity of cyclophosphamide resides with 4-hydroxycyclophosphamide and that its cytotoxic effect at therapeutic doses is largely mediated by phosphoramide mustard released within cells, these observations offer the possibility that the intracellular concentration of bifunctional catalysts, whether in the form of inorganic phosphates, organic phosphates, enzymes, or other species, serve as important determinants with regard to the oncotoxic potential and specificity of cyclophosphamide. Similarly, the concentration of bifunctional catalysis in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via acrolein, hemorrhagic cystitis.

Further studies on the conversion of 4-hydroxyoxazaphosphorines to reactive mustards and acrolein in inorganic buffers.

The rates at which the 4-hydroxyoxazaphosphorines, 4-hydroxycyclophosphamide and 4-hydroxyifosfamide, are converted to reactive mustards and acrolein in phosphate and bicarbonate buffers were determined as a function of pH, ionic strength, temperature, and buffer concentration. Conversion was first-order with respect to both the 4-hydroxyoxazaphosphorine and phosphate or carbonate serving as a catalyst. The catalytic constant for dianionic phosphate-catalyzed conversion of 4-hydroxyifosfamide to isophosphoramide mustard and acrolein at 37 degrees was 0.189 min-1; a value of 0.194 min-1 M-1 was obtained when dianionic phosphate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was examined. A catalytic constant of 3.09 min-1 M-1 was obtained for carbonate-catalyzed conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein. Hydroxyl ion and water also catalyzed the reaction; catalytic constants were 2710 and 0.000006 min-1 M-1, respectively. The rate at which the 4-hydroxyoxazaphosphorines were converted to reactive mustards and acrolein in phosphate buffer increased as the pH, ionic strength, and temperature increased. The energy of activation was about 20 kcal/mol. Dianionic phosphate, carbonate, hydroxyl ion, and water were apparently acting as general base catalysts of the rate-limiting step (probably the conversion of the intermediate aldehyde to the corresponding reactive mustard and acrolein) of the overall reaction, although specific base-general acid catalysis could not be ruled out. Bifunctional catalysis of the rate-limiting step did not appear to be significant and certainly was not obligatory as concluded previously by our laboratory. Assuming that the oncotoxic specificity of the oxazaphosphorines resides with the 4-hydroxyoxazaphosphorines and that their cytotoxic effect at therapeutic doses is largely mediated by the reactive mustards released within cells, these observations offer the possibility that intracellular general base catalytic activity serves as an important determinant with regard to the oncotoxic potential and specificity of the oxazaphosphorines. General base catalytic activity varies directly with pH, ionic strength, temperature, and the concentration of the base. The influence of some of these factors on the development of cyclophosphamide-induced bladder toxicity has already been demonstrated.

Effect of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and malignant blood cells to oxazaphosphorines.

The ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and several cultured human malignant blood cell lines to analogues of "activated" cyclophosphamide, namely, 4-hydroperoxycyclophosphamide and mafosfamide, and to phosphoramide mustard was quantified with and without concurrent exposure to an inhibitor of aldehyde dehydrogenase activity, namely, disulfiram, cyanamide, diethyldithiocarbamate, or ethylphenyl(2-formylethyl)phosphinate. Inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of 4-hydroperoxycyclophosphamide and mafosfamide toward all of the hematopoietic progenitors; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Potentiation of the cytotoxic action of mafosfamide toward cultured human malignant blood cells was minimal. Spectrophotometric assay revealed little NAD-linked aldehyde dehydrogenase activity present in the cultured human tumor cell lines as compared to that found in normal mouse liver or oxazaphosphorine-resistant L1210 cells. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major intermediate in cyclophosphamide bioactivation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that human multipotent hematopoietic progenitor cells contain the relevant aldehyde dehydrogenase activity, the relevant activity is retained upon differentiation to progenitors committed to the megakaryocytoid, granulocytoid/monocytoid, and erythroid lineages, and the relevant activity may be lost or diminished upon transformation of hematopoietic progenitors to malignant cells.

Cellular levels of class 1 and class 3 aldehyde dehydrogenases and certain other drug-metabolizing enzymes in human breast malignancies.

Molecular determinants of cellular sensitivity to cyclophosphamide, long the mainstay of chemotherapeutic regimens used to treat metastatic breast cancer, include class 1 and class 3 aldehyde dehydrogenases (ALDH-1 and ALDH-3, respectively), which catalyze the detoxification of this agent. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses that are obtained when such regimens are used to treat these malignancies. Consistent with this notion, ALDH-1 levels in primary and metastatic breast malignancies were found to range from 1-276 and 8-160 mIU/g tissue, respectively, and those of ALDH-3 range from 1-242 and 6-97 mIU/g tissue, respectively. ALDH-1 and ALDH-3 levels in normal breast tissue predicted the levels of these enzymes in primary and metastatic breast malignancies present in the same individuals. Confirming and extending the observations of others, levels of glutathione, a molecular determinant of cellular sensitivity to various DNA cross-linking agents including cyclophosphamide, and of DT-diaphorase, glutathione S-transferases, and cytochrome P450 1A1, each of which is known to catalyze the detoxification/toxification of one or more anticancer agents (although not of cyclophosphamide), also varied widely in primary and metastatic breast malignancies. Given the wide range of ALDH-1, ALDH-3, and glutathione levels that were observed in malignant breast tissues, measurement of their levels in normal breast tissue and/or primary breast malignancies prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack thereof, of cyclophosphamide in the treatment of metastatic breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens when treating this disease.

Identification of a class 3 aldehyde dehydrogenase in human saliva and increased levels of this enzyme, glutathione S-transferases, and DT-diaphorase in the saliva of subjects who continually ingest large quantities of coffee or broccoli.

Human saliva was tested for the presence of cytosolic class 3 aldehyde dehydrogenase, glutathione S-transferases alpha, mu, and pi, and DT-diaphorase, enzymes that are known to catalyze the biotransformation of many xenobiotics, including some that are carcinogens and some that are antineoplastic agents. Each of these enzymes was found to be present in this fluid. Inducers of these enzymes are known to be abundantly present in the human diet, especially in certain vegetables and fruits. Further investigation revealed that the salivary content of these enzymes rapidly, coordinately, and markedly increased upon daily consumption of relatively large amounts of coffee or broccoli. The enzyme activities of interest rapidly returned to basal levels when these substances were removed from the diet. Given the important role that cytosolic class 3 aldehyde dehydrogenase, the glutathione S-transferases, and DT-diaphorase are thought to play in determining the carcinogenic potential of some cancer-producing agents as well as the cytotoxic potential of some antineoplastic agents, and assuming that their salivary levels reflect their tissue levels, quantification of the salivary content of one or more of these enzymes, a noninvasive and relatively easy undertaking, could be useful in: (a) preliminarily assessing the chemopreventive potential of various diets and drugs; (b) establishing the optimal dose and schedule in Phase I clinical trials for any putatively chemopreventive diets or drugs of interest; and (c) the rational selection and use of chemotherapeutic agents, since several are inactivated, and a few are activated, by these enzymes; alternatively, the antineoplastic agent could be selected first and then a diet that enables the agent to achieve its full therapeutic potential would be selected based on whether high or low enzyme activity would be favorable in that regard. Such measurements may also be useful as an indicator when exposure to carcinogenic/teratogenic/otherwise toxic environmental/industrial/dietary agents that induce these enzymes is suspected.

Aldehyde dehydrogenase-mediated cellular relative insensitivity to the oxazaphosphorines.

As judged by findings in preclinical models, determinants of cellular sensitivity to cyclophosphamide and other oxazaphosphorines include two cytosolic aldehyde dehydrogenases, viz., ALDH1A1 and ALDH3A1. Each catalyzes the detoxification of the oxazaphosphorines; thus, cellular sensitivity to these agents decreases as cellular levels of ALDH1A1 and/or ALDH3A1 increase. Of particular clinical relevance may be that stable sublines, relatively insensitive to the oxazaphosphorines due to elevated ALDH1A1 or ALDH3A1 levels, emerged when cultured human tumor cells were exposed only once to a high concentration of one of these agents for 30 to 60 minutes. Whether differences in cellular levels of either enzyme accounts for the clinically-encountered uneven therapeutic effectiveness of the oxazaphosphorines remains to be determined. However, it has already been established that measurable levels of these enzymes are found in some, but not all, tumor types, and that in those tumor types where measurable levels are present, e.g., infiltrating ductal carcinomas of the breast, they vary widely from patient to patient. Potentially useful clinical strategies that might be pursued if it turns out that ALDH1A1 and/or ALDH3A1 are, indeed, clinically operative determinants of cellular sensitivity to the oxazaphosphorines include 1) individualizing cancer chemotherapeutic regimens based, at least in part, on the levels of these enzymes in the malignancy of interest, and 2) sensitizing tumor cells that express relatively large amounts of ALDH1A1 and/or ALDH3A1 to the oxazaphosphorines by preventing the synthesis of these enzymes, e.g., with antisense RNA, or by introducing an agent that directly inhibits the catalytic action of the operative enzyme. Further, the fact that ALDH1A1 and ALDH3A1 are determinants of cellular sensitivity to the oxazaphosphorines provides the rationale for the investigation of two additional strategies with clinical potential, viz., decreasing the sensitivity of vulnerable and essential normal cells, e.g., pluripotent hematopoietic cells, to the oxazaphosphorines by selectively transferring into them the genetic information that encodes 1) ALDH1A1 or ALDH3A1, or 2) a signaling factor, the presence of which would directly or indirectly, stably upregulate the expression of these enzymes.

Sensitivity of murine B- and T-lymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards.

The relative sensitivities of murine B- and T-lymphocytes to the oxazaphosphorine nitrogen mustards, cyclophosphamide and ASTA Z 7557, and to the non-oxazaphosphorine nitrogen mustards, melphalan and chlorambucil, in vivo, were determined. B- and T-lymphocytes were defined by selective mitogen-induced proliferation. Lipopolysaccharide (LPS)-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic effects of cyclophosphamide and ASTA Z 7557 as were phytohemagglutinin (PHA)- and concanavalin A (Con A)-induced T-lymphocytes. LPS-induced B-lymphocytes and PHA-induced T-lymphocytes were approximately equisensitive to the cytotoxic action of melphalan and chlorambucil, but the former were somewhat more sensitive to these agents than were Con A-induced T-lymphocytes. The relative sensitivities of murine B- and T-lymphocytes to ASTA Z 7557 and the non-oxazaphosphorine metabolite of cyclophosphamide, phosphoramide mustard, ex vivo, were also determined. LPS-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic action of ASTA Z 7557 as were PHA- and Con A-induced T-lymphocytes. The three mitogen-induced lymphocyte populations were approximately equisensitive to the cytotoxic action of phosphoramide mustard. These observations suggest that the differential effect of cyclophosphamide on murine B- and T-lymphocytes is uniquely exhibited by oxazaphosphorine nitrogen mustards. Furthermore, the results suggest that 4-hydroxycyclophosphamide is the cyclophosphamide metabolite that mediates the differential immunotoxic effect of the parent compound.

Aldehyde dehydrogenase activity as the basis for the relative insensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines.

The ex vivo sensitivity of murine pluripotent hematopoietic stem cells (CFU-S) and myeloid progenitor cells (CFU-GM) to 4-hydroperoxycyclophosphamide, ASTA Z 7557, phosphoramide mustard, acrolein, melphalan, and cis-platinum was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. As compared to CFU-GM, CFU-S were less sensitive to the oxazaphosphorine agents, 4-hydroperoxycyclophosphamide and ASTA Z 7557. The two cell populations were approximately equisensitive to acrolein as well as to the non-oxazaphosphorine cross-linking agents, phosphoramide mustard, melphalan and cis-platinum. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines toward CFU-S; they did not potentiate the cytotoxic action of acrolein or the non-oxazaphosphorines toward these cells. The inhibitors did not potentiate the cytotoxic action of the oxazaphosphorines, non-oxazaphosphorines, or acrolein toward CFU-GM. Pyridoxal, a substrate for aldehyde oxidase, did not potentiate the cytotoxic action of oxazaphosphorines toward CFU-S. Cellular NAD-linked aldehyde dehydrogenases are known to catalyze the oxidation of the major transport form of cyclophosphamide, 4-hydroxycyclophosphamide/aldophosphamide, to an inactive metabolite, carboxyphosphamide. Our observations suggest that (1) aldehyde dehydrogenase activity is an important determinant of the sensitivity of a cell population to the oxazaphosphorines, (2) CFU-GM lack the relevant aldehyde dehydrogenase activity, and (3) the phenotypic basis for the relative insensitivity of CFU-S to oxazaphosphorines is the aldehyde dehydrogenase activity contained by these cells.

Influence of pH on the cytotoxic activity of chlorambucil.

The cytotoxic activity of chlorambucil as a function of pH was investigated in P388 tumor cells growing in static suspension culture. A decrease in extracellular pH from 7.8 to 7.2 was associated with a decrease in intracellular pH from 7.92 to 7.55. The cytotoxic potency of chlorambucil increased as the extracellular pH decreased; IC99 values were 20 and 60 microM when the extracellular pH was 7.2 and 7.8 respectively. Covalent binding to cellular macromolecules was about 1.9 times greater at pH 7.2 relative to that at pH 7.8. These results suggest that pH may be an important determinant of the oncotoxic specificity of chlorambucil, and that the cytotoxic activity of this agent could be selectively directed toward tumor cells by the selective manipulation of intracellular and extracellular pH. A potential influence of intracellular and extracellular pH on cytotoxic, mutagenic, carcinogenic, and teratogenic potencies of other chemicals is also suggested. Additionally, these investigations demonstrate the importance of carefully controlling pH throughout the drug exposure period when evaluating the relative potency of potential cytotoxic, mutagenic, carcinogenic, and teratogenic agents in cell or organ culture.

Identification of the class-3 aldehyde dehydrogenases present in human MCF-7/0 breast adenocarcinoma cells and normal human breast tissue.

Affinity column chromatography was used to semipurify the very small amounts of class-3 aldehyde dehydrogenase (ALDH-3) present in human MCF-7/0 breast adenocarcinoma cells and human normal breast tissue. Characterization of the semipurified enzymes revealed that each was a type-1 ALDH-3 rather than a type-2 ALDH-3 as previously reported. Although clearly a type-1 ALDH-3, the MCF-7/0 enzyme, as well as the type-1 ALDH-3 constitutively present in cultured colon C cells and induced in cultured MCF-7/0 cells by methylcholanthrene, does, however, differ from the prototypical human stomach mucosa type-1 ALDH-3 in one, perhaps pharmacologically important, way, viz. when the ability to catalyse the oxidation of aldophosphamide is normalized by the ability to catalyse the oxidation of benzaldehyde, each of these enzymes, as well as the type-2 ALDH-3 found in MCF-7/OAP cells, exhibits greater ability to catalyse the oxidation of aldophosphamide than does stomach mucosa type-1 ALDH-3; hence, although not type-2 ALDH-3s, they may be slight variants of the prototypical type-1 ALDH-3.

Kinetic characterization of the catalysis of "activated" cyclophosphamide (4-hydroxycyclophosphamide/aldophosphamide) oxidation to carboxyphosphamide by mouse hepatic aldehyde dehydrogenases.

A spectrophotometric assay was developed and utilized to directly characterize aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide to carboxyphosphamide by soluble and solubilized particulate fractions prepared from mouse liver homogenates. Vmax values of 3310 and 1170 nmol/min/g liver were obtained for the soluble and solubilized particulate fractions respectively. Km values were 22 and 84 microM respectively. Alkaline pH optimums were observed in each case. Aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide by the soluble fraction was markedly more temperature responsive. Catalysis of aldophosphamide and acetaldehyde or benzaldehyde oxidation was apparently by the same isozyme(s) in the soluble fraction. Similarly, low Km (acetaldehyde/benzaldehyde) and high Km (acetaldehyde/benzaldehyde) isozymes each apparently catalyzed the oxidation of aldophosphamide in the solubilized particulate fraction. Our findings suggest that (1) oxidation of aldophosphamide to carboxyphosphamide by mouse liver is catalyzed largely by the predominant aldehyde dehydrogenase isozyme present in the soluble fraction (cytosol) of this tissue, and (2) isozymes that catalyze aldophosphamide oxidation are not different from those that catalyze the oxidation of acetaldehyde and benzaldehyde, though the relative contribution of each isozyme within the solubilized particulate fraction to the catalysis of aldophosphamide oxidation remains to be determined.

Sensitivity of aldehyde dehydrogenases in murine tumor and hematopoietic progenitor cells to inhibition by chloral hydrate as determined by the ability of chloral hydrate to potentiate the cytotoxic action of mafosfamide.

Several murine aldehyde dehydrogenases, most notably AHD-2, are known to catalyze the detoxification of cyclophosphamide, mafosfamide, and other oxazaphosphorines. Thus, cellular sensitivity to these agents decreases as the relevant aldehyde dehydrogenase activity increases, and vice versa. Chloral hydrate is a sedative/hypnotic agent that is sometimes administered to patients being treated with cyclophosphamide. It is known to inhibit some, but not all, aldehyde dehydrogenases. Murine (CFU-S, CFU-GEMM and CFU-Mk) and human (CFU-Mix, CFU-GM, BFU-E and CFU-Mk) hematopoietic progenitor cells, as well as murine oxazaphosphorine-resistant (L1210/OAP and P388/CLA) tumor cells, are known to contain the relevant aldehyde dehydrogenase activity but the identity of the specific enzyme present in the normal cells is unknown and may be different than that, namely AHD-2, present in neoplastic cells. In that event, the potential exists to inhibit the detoxification of the oxazaphosphorines in tumor cells without inhibiting this event in normal cells; the net effect of such a selective inhibition would be to increase the margin of safety of the oxazaphosphorines. In ex vivo experiments, chloral hydrate markedly potentiated the antitumor activity of mafosfamide against oxazaphosphorine-resistant L1210/OAP and P388/CLA cells. It did not potentiate the cytotoxic action of mafosfamide against any of the murine or human hematopoietic cells tested, even at concentrations which fully restored the sensitivity of the resistant tumor cell lines to this agent. One explanation for these observations is that hematopoietic progenitor, and the resistant tumor, cells express different relevant aldehyde dehydrogenases and that these aldehyde dehydrogenases differ in their sensitivity to inhibition by chloral hydrate. Consistent with this notion were the observations that AHD-2 was exquisitely sensitive to inhibition by chloral hydrate, whereas two other aldehyde dehydrogenases that also catalyze the detoxification of aldophosphamide, namely AHD-12a, b and AHD-13, were relatively unaffected.

NADPH-dependent enzyme-catalyzed reduction of aldophosphamide, the pivotal metabolite of cyclophosphamide.

One of the metabolites found in the urine of mammals given the prodrug cyclophosphamide is alcophosphamide, an alcohol. It is most probably generated from cyclophosphamide via aldophosphamide, an aldehyde which otherwise can directly give rise to phosphoramide mustard; the latter effects the cytotoxic action of cyclophosphamide and other oxazaphosphorines. It has already been demonstrated that horse liver alcohol dehydrogenase catalyzes the reduction of aldophosphamide to alcophosphamide. Herein, we report that aldose reductase and aldehyde reductase purified from human placenta also catalyze this reaction. The Km values for aldose reductase- and aldehyde reductase-catalyzed reduction of aldophosphamide to alcophosphamide were 0.15 and 1.6 mM, respectively. Aldose reductase and aldehyde reductase accounted for 94 and 6%, respectively, of total placental pyridine nucleotide-dependent enzyme-catalyzed aldophosphamide (160 microM) reduction. Aldose reductase-catalyzed reduction of aldophosphamide appeared to be noncompetitively inhibited by sorbinil; the Ki value was 0.4 microM. The in vivo significance of these observations is uncertain but could be of some magnitude since alcophosphamide is known to be only weakly cytotoxic.

Identification and characterization of a novel class 3 aldehyde dehydrogenase overexpressed in a human breast adenocarcinoma cell line exhibiting oxazaphosphorine-specific acquired resistance.

Associated with the oxazaphosphorine-specific acquired resistance exhibited by a human breast adenocarcinoma subline growing in monolayer culture, viz. MCF-7/OAP, was the overexpression (> 100-fold as compared with the very small amount expressed in the oxazaphosphorine-sensitive parent line) of a class 3 aldehyde dehydrogenase, viz. ALDH-3, judged to be so because it is a polymorphic enzyme (pI values ca. 6.0) present in the cytosol that is heat labile, is insensitive to inhibition by disulfiram (25 microM), much prefers benzaldehyde to acetaldehyde as a substrate and, at concentrations of 4 mM, prefers NADP to NAD as a cofactor. No other aldehyde dehydrogenases were found in these cells. As compared with those of the prototypical class 3 human ALDH-3, viz. constitutive human stomach mucosa ALDH-3, the physical and catalytic properties of the MCF-7/OAP enzyme differed somewhat with regard to pI values, native M(r), subunit M(r), recognition of the subunit by anti-stomach ALDH-3 IgY, pH stability, cofactor influence on catalytic activity, and the ability to catalyze, albeit poorly, the oxidation of an oxazaphosphorine, viz. aldophosphamide. Hence, the MCF-7/OAP ALDH-3 was judged to be a novel class 3 aldehyde dehydrogenase. Small amounts of a seemingly identical enzyme are also present in normal pre- and post-menopausal breast tissue. None could be detected in human liver, kidney or placenta, suggesting that it may be a tissue-specific enzyme.

Effects of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of murine late spleen colony-forming cells (day-12 CFU-S) and hematopoietic repopulating cells to mafosfamide (ASTA Z 7557).

The effects of inhibitors of aldehyde dehydrogenase activity on the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorine anticancer agents, e.g. mafosfamide, were examined using two different assay procedures. In the first part of the investigation, the ex vivo sensitivity of murine day-12 spleen colony-forming cells (CFU-S) to mafosfamide was determined in the absence and presence of known inhibitors of aldehyde dehydrogenase activity, viz. diethyldithiocarbamate and cyanamide. These results were compared to those generated for day-8 CFU-S. Day-12 CFU-S were less sensitive to mafosfamide, and to phosphoramide mustard, although the difference in sensitivity to the latter was less marked. Diethyldithiocarbamate and cyanamide each potentiated the cytotoxic action of mafosfamide toward both day-12 and day-8 CFU-S; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Since cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major transport form of mafosfamide, to the relatively nontoxic acid, carboxyphosphamide, the results suggest that intracellular aldehyde dehydrogenase activity is a determinant of the sensitivity of day-12 CFU-S, as well as of day-8 CFU-S, to mafosfamide and other oxazaphosphorines, e.g. cyclophosphamide. In the second part of this investigation, a murine syngeneic bone marrow transplantation model was used to determine the ex vivo sensitivity of murine hematopoietic repopulating cells to mafosfamide in the absence and presence of diethyldithiocarbamate. Specifically, the ability of treated marrow grafts to repopulate the hematopoietic system, and thereby save recipients from the otherwise lethal effect of total body irradiation, was determined. Diethyldithiocarbamate potentiated the cytotoxic action of mafosfamide, but not that of phosphoramide mustard, toward hematopoietic repopulating cells. These observations support our previous contention that aldehyde dehydrogenase activity is an operative determinant with regard to the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines.