Effects of sodium nitrite and potassium sorbate on in vitro cultured mammalian cells

The food additives sodium nitrite and potassium sorbate had cytostatic and cytotoxic effects on in vitro cultured V79 hamster cells and EUE human fibroblasts if administered in an acid environment (pH 4.95). The strong cytotoxic effect of sodium nitrite and that of the combined action of sodium nitrite and potassium sorbate was observed along the inhibition of macromolecular synthesis. In this respect, potassium sorbate was less effective. The decreased plating efficiency of the cells and the inhibition of de novo DNA synthesis induced by these substances aroused the question whether they also have genotoxic effects on V79 cells. Statistical analyses showed that sodium nitrite induced more 6-TG-resistant (6-TGr) mutants as compared to the untreated control. However, this elevation did not correspond to the level of inhibition of DNA synthesis determined during the followed period of time after the removal of the substance. Potassium sorbate and a combination thereof with sodium nitrite, in our experiments, had no mutagenic effects.     

Interaction of etoposide and cisplatin in an in vitro tumor model

Some clinical data suggest that etoposide and cisplatin may be synergistic against certain types of tumors. Since studies of the process are both easier and more precise in laboratory systems, we have used V79 spheroids as a model to quantify the interaction of the drugs in cycling and noncycling, aerobic, and hypoxic cell subpopulations. The degree of interaction between etoposide and cisplatin in this system, quantified using the median-effect analysis, was highly dependent on the overall treatment toxicity, and to a lesser extent, the subpopulation of cells assessed. Synergism was demonstrated for relatively modest (1 decade) cell kills achievable at clinically relevant drug doses; increasing the toxicity of the combination treatment by increasing the cisplatin level produced a greater relative gain in the noncycling and hypoxic cells than in the aerobic, cycling cells of the spheroids.     

Lactate metabolism in fetal type II pneumocytes

Fetal rat lung type II pneumocytes in organotypic culture produce significant quantities of both pyruvate and lactate, even when maintained under aerobic conditions (5% CO₂/air). A hypoxic atmosphere of 95% nitrogen/5% CO₂ increased lactate production by 35%, while cells in a hyperoxic environment (95% oxygen/5% CO₂) manufactured lactate at a rate similar to controls. Cells incubated under aerobic conditions following hypoxia synthesized lactate at a rate intermediate between those found for the aerobic and hypoxic states. The presence of 1.5 mM glucose in the incubation medium resulted in a 79% increase in lactate synthesis, but no further increases were found with extracellular glucose concentrations up to 20 mM. Lactate and pyruvate production by type II cells was decreased in a concentration-dependent fashion by the presence of these metabolites in the incubation medium. Physiological levels of pyruvate or lactate caused a net utilization of these substrates by the type II pneumocyte. These data indicate that fetal lung type II pneumocytes are capable of producing pyruvate and lactate under aerobic conditions and that these cells are apparently able to adjust the rate of lactate production to the extracellular oxygen tension. This study also suggests that the type II pneumocyte may be a net utilizer of lactate and pyruvate under the conditions present in utero during late gestation.     

Identification of 7,12-dimethylbenz[a]anthracene metabolites that lead to mutagenesis in mammalian cells

The mutagenicity of 7,12-dimethylbenz[a]-anthracene (DMBA) and 11 of its enzymatically derived metabolites was tested with Chinese hamster V79 cells for identification of mutagenic metabolites. The metabolites consisted of 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, three trans-3,4-diols, two trans-5,6-diols, and three trans-8,9-diols, all of which derived from DMBA or from the hydroxymethyl derivatives. Mutations were characterized by resistance to ouabain and 6-thioguanine. None of the tested metabolites were mutagenic in V79 cells, which do not metabolize polycyclic aromatic hydrocarbons. Therefore, mutagenesis in the V79 cells was tested in the presence of golden hamster cells capable of metabolizing polycyclic aromatic hydrocarbons (cell-mediated assay). In this assay, DMBA, 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]anthracene, and their trans3,4-diols were mutagenic for both genetic markers, and the mutagenic response increased as a function of the hydrocarbon dose. All other metabolites were either inactive or showed up to a 4-fold higher mutation frequency than the untreated V79 cells for ouabain and 6-thioguanine resistance. The DMBA-trans-3,4-diol was the only metabolite that was more active than DMBA itself; at 0.05 muM it was 6-8 times more active than DMBA itself; at 0.05 muM it was 6-8 times more active than DMBA in inducing both ouabain and 6-thioguanine resistance. This diol was mutagenic at a dose as low as 0.01 muM. Mutagenesis by DMBA and the trans-3,4-diols was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Analysis of DMBA metabolism in intact golden hamster cells indicated that DMBA-trans-3,4-diol is one of the major metabolites produced. Our results therefore suggest that DMBA-trans-3,4-diol may be metabolized to a diol-epoxide, presumably the trans-3,4-diol-1,2-epoxide, which may be a major reactive metabolite responsible for DMBA mutagenicity in mammalian cells.     

Pre-screening of carcinogens by studying of DNA synthesis inhibition and gene mutations in mammalian cells

The ability to induce gene mutations at the HGPRT locus in Chinese hamster V79 cells and to inhibit DNA synthesis in human EUE cells has been followed in eighteen chemical substances and ultraviolet light. The aim was to determine whether following of inhibition of DNA synthesis in impaired human cells may serve as an indicator of the mutagenicity of the relevant chemical substance. The results permit us to assume that following of DNA synthesis, the so-called DNA-inhibition test provides a preliminary information on the genotoxicity of the chemical substance, although the degree of inhibition of DNA synthesis need not necessarily denote the measure of the mutagenic effect of the substance. As certain further tests, utilising in vitro mammalian cells, so also the DNA-inhibition test yields a certain percentage of false positive results. Hence, it is important to combine this test with further express in vitro tests on the level of mammalian cells.     

Differences in mutagenicity and cytotoxicity of (+)- and (-)-benzo[a]pyrene 4,5-oxide: a synergistic interaction of enantiomers.

In order to study the biological effects of (+)- and (-)-benzo[a]pyrene 4,5-oxide, a synthesis of these molecules has been developed based on the resolution of (+/-)-cis-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene. The (-) enantiomer of benzo[a]pyrene 4,5-oxide was 1.5- to 5.5-fold more mutagenic than the (+) enantiomer in strains TA 98, TA 100, TA 1537, and TA 1538 of Salmonella typhimurium and in Chinese hamster V79 cells. In studies with V79 Cells, the (-) enantiomer of benzo[a]pyrene 4,5-oxide was also more cytotoxic than the (+) enantiomer. When mixtures of the enantiomers were studied in V79 cells, synergistic cytotoxic and mutagenic responses were observed. The greatest cytotoxic and mutagenic effects occurred with a 3:1 mixture of the (-) and (+) enantiomers of benzo[a]pyrene 4,5-oxide, respectively.     

Sectional analysis of tumor colony growth in the soft-agar assay: Effects of oxygen

Summary Soft-agar clonogenicity of L1210 mouse leukemia cells and of xenografts of a human melanoma and a carcinoma of the cervix was studied sectionally by the sizes of the colonies grown under hypoxic gradients and aerobic condition. Soft-agar plating efficiency was increased in cultured L1210 cells with decreasing oxygen concentrations. The growth of both cultured L1210 cells and their BDF1 ascites was better in 5% oxygen than in 20% oxygen. Although soft-agar colony development of both melanoma and cervical carcinoma was significantly better in 5% oxygen, the former has a secondary preference for a hypoxic atmosphere and the latter, for an aerobic condition.Abbreviation PE plating efficiency This work was supported in part by American Cancer Society Grants #IN-51V and #IN-150, and by a Cancer Research Grant from the Elsa U. Pardee Foundation, Midland, Mich, USA     

Effect of glycolytic inhibitors on the sinoatrial node,atrium and atrioventricular node in the rabbit heart

The effect of unspecific noncompetitive (iodoacetic acid; IAA) and of specific competitive (deoxyglucose) glycolytic inhibitors were studied under aerobic and anaerobic conditions in the isolated sinoatrial (SA) node, right atrium and atrioventricular (AV) node of the rabbit heart. Transmembrane action potentials were recorded simultaneously with the His bundle electrogram. Under aerobic conditions (Po₂ 460 mmHg), 7.5 × 10^?5m IAA caused no significant alterations of sinus rate, intraatrial and AV nodal conduction. Under hypoxic conditions (Po₂ 40 mmHg), 7.5 × 10^?5m IAA resulted in rapid and complete abolition of the SA and AV nodal electrical activity and in a more delayed atrial inexcitability. Similar changes were never observed in the presence of hypoxia alone. Higher concentrations of IAA (5 × 10^?4m) severely depressed the sinus rate and the AV nodal conduction already under aerobic conditions. The action potential amplitude and the rate of diastolic depolarization of the SA nodal fibers was significantly reduced, the maximum diastolic potential remained unchanged. In atrial fibers, 5 × 10^?4m IAA caused predominantly shortening of the action potential duration but had less marked and delayed depressant effects on the action potential amplitude. Electrophysiologic abnormalities included Mobitz type II sinoatrial block. The electrophysiological effects of deoxyglucose (50 mm) under aerobic and hypoxic conditions resembled those obtained with 7.5 × 10^?5m IAA. The results suggest that the “normal” nodal function is predominantly related to aerobic metabolism but that under hypoxic conditions glycolytic energy production can effectively contribute to the maintenance of nodal electrical activity. Also that the specific relation between cardiac metabolism and electrical activity of nodal and atrial cells may at least partly be explained by the particular electrical membrane properties of the various fiber types.     

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme.     

Liver cell-mediated mutagenesis of mammalian cells by liver carcinogens.

A cell-mediated mutagenesis assay using primary cultures of rat liver cells and V79 Chinese hamster cells has been developed. Liver carcinogens and their structural analogues were studied. Mutations in the V79 cells were characterized by resistance to ouabain. Cocultivation of the liver cells and V79 cells in the presence of the carcinogens N-nitrosodimethylamine, N-nitrosodiethylamine, and aflatoxin B1 caused the induction of ouabain-resistant mutants of V79 cells. In the absence of liver cells, the carcinogens did not induce ouabain resistance. The analogues N-nitrosomethyl-tert-butylamine and aflatoxin G2 were not mutagenic. The carcinogens exhibited a dose-dependent enhancement of mutation frequency. The mutation frequency also increased with increasing numbers of liver cells seeded. It is suggested that such an experimental system may be useful for screening for chemical carcinogens.     

Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells.

The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.     

Comparative studies on fatty acid synthesis in atherosclerotic and hypoxic human aorta.

The oxygen and glucose uptake, lactate formation, ATP/ADP and NADH/NAD ratios and incorporation of [14C]acetate and [14C]linolenic acid into lipids of early fatty streaks and more advanced complicated atherosclerotic lesions of human aorta were determined during aerobic and hypoxic incubation. Compared with grossly normal appearing sections of the aorta in intima and media preparations of early fatty streaks the oxygen uptake was increased while that in further developed atheroma was slightly diminished. Under aerobic incubation conditions the metabolic state of fatty streaks and atheroma was characterized by increased lactate formation, NADH/NAD ratio and incorporation of [14C]acetate and [14C]linolenic acid into the lipids, but by a lowered ATP/ADP ratio. More pronounced changes in these metabolic parameters were observed when the aortic tissue segments were incubated under hypoxic conditions. The analysis by argentation TLC of fatty acid methylesters derived from total lipids of aerobically incubated fatty streaks revealed an increased incorporation of [14C]acetate into the highly unsaturated long-chain fatty acids. In developed atherosclerotic lesions and in hypoxia the incorporation of radioacetate into the polyunsaturated fatty acids and the formation of 20:4 fatty acid from [14C]linolenic acid were, in contrast to the above finding, decreased while the synthesis of eicosatrienoic acid was increased. This finding suggests a block in the desaturation step of linoleic into 20:4 fatty acid in further developed atheroma and in hypoxia. In aerobically incubated atherosclerotic lesions and in hypoxia the palmitic acid was synthesized mainly by chain elongation while in grossly normal areas of the aorta at least part of this acid was synthesized de novo.     

Results of genotoxicity testing of theophylline on bacteria and two lines of mammalian cells

In study of the genotoxic effects of theophylline, this substance was subjected to a series of tests. Its potential mutagenicity was followed at the level of both bacteria and mammalian cells. The capacity of the substance to damage human DNA was determined by the so-called DNA inhibition test and by the method of alkaline elution of DNA. In the absence of the enzymatic microsomal S9 fraction, theophylline showed very weak mutagenic effects on bacteria and mammalian cells. However, in both cases this weak mutagenic effect was eliminated through a simultaneous application of theophylline and the S9 fraction. The results of the remaining tests proved negative regardless of whether the S9 fraction was present or absent. Our results lead us to infer that theophylline exerts no genotoxic action under in vivo conditions.     

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.Changes in cancer-cell metabolism have been linked to genetic alterations of oncogenes, tumor suppressors, and metabolic enzymes (1–3). The hypoxic tumor microenvironment further modifies metabolism through activation of hypoxia-inducible factors (HIFs). The HIFs enhance tumorigenesis by stimulating glycolysis, cell motility, and angiogenesis (4, 5). Thus, hypoxia portends poor prognosis in common cancers, such as gastric, lung, ovarian, pancreatic, prostate, and renal carcinomas (5).Although Otto Warburg observed respiration in certain cancer types, his obsession with aerobic glycolysis as a cause of cancer promulgated the prevailing misconception that cancers only exhibit the Warburg effect exclusive of respiration (6). Because the hypoxic tumor microenvironment activates HIFs and diminishes respiration, whether hypoxia enhances tumorigenicity at the expense of respiration is not fully understood (7). We found recently that oxidative and glycolytic metabolism coexist in hypoxic B lymphocytes, such that the shunting of glucose to lactate away from the tricarboxylic acid cycle (TCA) cycle by hypoxia is compensated through glutamine oxidation in the TCA cycle (8). These metabolic aberrations suggest the existence of hypoxic respiring (herein termed non-Warburg) cells capable of continued oxidative metabolism under hypoxic conditions. Further, it is believed that cancer cells within the tumor microenvironment are either aerobic or hypoxic because of oxygen gradients coming from nearby imperfect blood vessels. An intriguing commensal metabolic relationship between hypoxic and aerobic cells has been documented, whereby hypoxic cells produce lactate that is converted to pyruvate for respiration by aerobic cancer cells located nearby the blood vessel (9). We hypothesize, however, that hypoxic respiring cells could also participate in this commensal relationship, specifically because oxygen becomes limiting for cytochrome c oxidase and cellular respiration only around 0.1% and 0.5% oxygen, respectively (10). In this regard, we sought to determine the gene expression and tumorigenic phenotypes of these putative non-Warburg cells and other hypoxic tumor-cell subpopulations. Toward this end, we developed a reporter system that identifies hypoxic and/or cycling cells.We constructed a dual fluorescent protein reporter system—hypoxia and cell cycle reporter (HypoxCR)—that simultaneously detects hypoxic and/or dividing cells. Using HEK293T cells stably expressing HypoxCR as a model, we identified and purified four distinct hypoxic cell populations by flow cytometry. We surmise that these four populations reflect the heterogeneity of the solid tumor microenvironment that we observed by microscopy in HEK293T xenografts. Each hypoxic cell subpopulation has distinct gene-expression profiles. The population that was HIF-negative and noncycling had increased expression of mitochondrial genes. As a purified population, these non-HIF/noncycling cells also had the highest oxygen-consumption rate and mitochondrial capacity. Surprisingly, we found that these cells were tumorigenic in xenografts, similar to the cells that were HIF-positive and cycling. Neither HIF-positive noncycling cells nor cycling HIF-negative cells were capable of establishing tumor xenografts. Furthermore, we provide proof-of-concept studies for the use of HypoxCR in vivo and found that bevacizumab (VEGF pathway inhibitor) increased the HIF-positive cell population, consistent with a vascular pruning effect.     

Modulation of sensitivity to mitomycin C and a dithiol analogue by tempol in non-small-cell lung cancer cell lines under hypoxia

We examined the mechanisms involved in the bioactivation of mitomycin C (MMC) and a newly developed MMC analogue: 7-N-(2-{[2-(-l-glutamylamino)ethyl]dithio}ethyl)mitomycin C, KW-2149, in non-small-cell lung cancer (NSCLC) cell lines under aerobic and hypoxic conditions. To investigate these mechanisms, we used MMC-resistant non-small-cell lung cancer cell lines (PC-9/MC4) that had been established in our laboratory from the parent PC-9 cell line by continuous exposure to MMC. We previously reported that the MMC-resistant cell line (PC-9/MC4) was poor in NAD(P)H dehydrogenase (quinone) activity and approximately 6-fold more resistant than the parent cells (PC-9) to MMC on 2-h exposure under aerobic conditions. In this study, the subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9, the parent cell line, under aerobic conditions, and 5.2-fold more resistant under hypoxic conditions after 2h exposure to MMC. However, on co-incubation with tempol, an inhibitor of the one-electron reduction pathway, the sensitivity of PC-9/MC4 to MMC was impaired under hypoxic conditions, but the impairment was not evident under aerobic conditions. KW-2149, the newly developed MMC analogue, was cytotoxic for both PC-9/MC4 and PC-9 cells, and the sensitivity of both cell lines to KW-2149 was not changed by exposure to hypoxic conditions or by coincubation with tempol. There were no significant differences in the intracellular uptake of MMC and the activities of cytosolic detoxification enzymes between the PC-9 and PC-9/MC4 cell lines. These results support the hypothesis that the one-electron reduction pathway plays a partial role in the bioactivation of MMC, but not of KW-2149, and that KW-2149 is excellent at circumventing resistance to MMC in NSCLC.Abbreviations KW-2149 7-N-(2-{[2-(-l-glutamylamino)ethyl]dithio}ethyl)mitomycin C - MMC mitomycin C - NSCLC non-small-cell lung cancer - PBS calcium and magnesium-free Dulbecco's phosphate-buffered saline - P-450 reductase NADPH-cytochromeP-450 reductase     

Occurrence of induced 6-thioguanine-resistant colonies in synchronized V79 cells after treatment with ftorafur. Effects of the S9 fraction

The capacity of ftorafur to induce resistance to 6-thioguanine in V79 hamster cells was investigated. The treatment conditions were arranged in order to favor the induction of such gene mutations. The synchronous cells were treated in the beginning of S phase, i.e., when the replication of the genes responsible for the expression of resistance to 6-thioguanine took place. Part of the experiments was carried out in presence of the S9 microsomal fraction. The results showed that ftorafur was a weak mutagen after a short-term exposure in the presence of adequate amounts of the S9 fraction. In absence of the S9 fraction or when other than-optimal amounts were used, we repeatedly failed to detect any increase in the occurrence of mutated cells. Also the cytotoxic effects of this substance were investigated. We found that a short-term treatment of V79 hamster cells with lower concentrations of ftorafur resulted in decrease of growth rate, higher concentrations after a short-term treatment were in turn slightly toxic. Long-term treatments are, depending on the concentration and time of treatment used, considerably toxic.     

Stimulated synthesis of prostaglandin E2 or leukotrien C4 from myocardial cells is not a cause but a result of their injury under hypoxia

Cardiac muscle cell injury may be related to metabolic changes associated with a rise in intracellular calcium. The mechanisms by which an elevated Ca2+ can cause injury are uncertain, but injury could occur by activation of any one or several calcium-dependent processes. To examine whether the process is mediated by prostaglandins (PG) or leukotriens (LT), we measured the successive release of creatine kinase (CK), PGE2 and LTC4 that have been reported to induce the cell injury via the arachidonic acid cascade, to the culture medium from myocardial cells under hypoxic or aerobic conditions. CK release, a biochemical marker of muscle cell necrosis, was first detected in the medium of hypoxic cultures at 9 h. Both PGE2 and LTC4 production and release were delayed, being first detected at 12 h after initiating hypoxia treatment. Addition of exogenous PGE2 or LTC4 to the culture medium (1.0 or 10 ng/ml) did not cause any effect on the CK release under aerobic condition. Cyclooxygenase inhibitor, indomethacin (1 X 10(-5) M) or lipoxygenase inhibitor, AA861 (1 X 10(-5) M), reduced the synthesis of PGE2 by 80% or LTC4 by 68% under hypoxia, respectively, but caused no beneficial effect on the CK release. These findings suggest that cardiac muscle cells themselves produce PGE2 and LTC4 after hypoxia and that the production of these compounds merely occurs as a result, but not as a cause of cell injury.     

Mutagenic and cytotoxic activity of benzol[a]pyrene 4,5-, 7,8-, and 9,10-oxides and the six corresponding phenols.

The benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides and the six corresponding phenols (4-, 5-, 7-, 8-, 9-, and 10-hydroxybenzo[a]pyrene) have been tested for mutagenic and cytotoxic activity in bacteria and in a mammalian cell culture system. Benzo[a]pyrene 4,5-oxide (K-region) was highly mutagenic in two histidine-dependent strains (TA1537 and TA1538) of Salmonella typhimurium which detect frameshift mutagens. In contrast, benzo[a]pyrene 7,8- and 9,10-oxides were less than 1% as mutagenic as the 4,5-oxide. Benzo[a]pyrene 7,8- and 9,10-oxides were unstable in aqueous media, whereas the 4,5-oxide was stable for several hours. This difference in stability could not account for the different mutagenic activities of the three arene oxides. The benzo[a]pyrene oxides were inactive in a strain (TA1535) that is reverted by base pair mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine or in a strain (TA1536) that detects framshift mutagens similar to the acridine half-mustard ICR-191. Benzo-[a]-pyrene and the six phenols were all stable in aqueous media, but they had little or no mutagenic activity in any of the four Salmonella strains. Conversion of 8-azaguanine-sensitive Chinese hamster V79 cells to 8-azaguanine-resistant variants was increased by benzo[a]pyrene 4,5-oxide, whereas the 9,10-oxide was considerably less active. Benzo[a]pyrene and the other derivatives had little or no effect. Benzo[a]yrene 4,5-oxide was more cytotoxic to the Chinese hamster V79 cells than the 7,8- and 9,10-oxides, while 8-hydroxybenzo[a]pyrene was the most cytotoxic of the six phenols.     

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.