Diaziquone-induced cytotoxicity in isolated rat hepatocytes

2,5-Diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone (AZQ) is a lipid-soluble antitumor agent. The following evidence using isolated rat hepatocytes as a model for cytotoxicity studies suggests that, under aerobic conditions, AZQ participates in futile oxidation-reduction cycling and oxygen activation. The H2O2 formed can mediate oxidative stress cytotoxicity in compromised cells. (a) Addition of AZQ to hepatocytes causes the stoichiometric oxidation of glutathione (GSH) to oxidized glutathione. No subsequent reduction back to GSH occurred. This was found to be the result of reversible inactivation of glutathione reductase by AZQ. The extent of GSH oxidation increased with AZQ concentration. (b) Cytotoxicity occurred when AZQ concentrations were sufficient to completely deplete GSH. (c) Addition of AZQ to hepatocytes enhanced cyanide-resistant respiration. (d) If the hepatocytes were compromised with azide or cyanamide to inhibit catalase, cytotoxicity was increased 10-fold or 100-fold if ascorbate was also present. (e) AZQ readily induced Ca2+ release by energized mitochondria. Ascorbate markedly enhanced the effectiveness of AZQ, and catalase delayed Ca2+ release. H2O2 formed by aerobic oxidation-reduction cycling of AZQ may therefore cause mitochondrial Ca2+ release.     

Molecular mechanisms of SR 4233-induced hepatocyte toxicity under aerobic versus hypoxic conditions.

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is the lead compound of the benzotriazene-di-N oxides which are selectively toxic to tumour cells under hypoxic conditions. However much higher concentrations given to rats caused bone marrow toxicity and necrosis of the low oxygen Zone 3 part of the liver. In the following effects of SR 4233 on hepatocytes under hypoxic vs aerobic conditions have been compared. (1) SR 4233 did not affect hepatocyte viability (as determined by plasma membrane disruption) or glutathione levels under aerobic conditions. SR 4233 however induced cyanide-resistant respiration, an indicator of redox cycling mediated oxidative stress and became cytotoxic if hepatocyte catalase or glutathione reductase was inactivated. Glutathione oxidation occurred well before cytotoxicity ensued. Addition of ascorbate markedly enhanced SR 4233 cytotoxicity to these compromised hepatocytes. (2) In contrast, SR 4233 was highly toxic to hypoxic hepatocytes. Addition of ascorbate to enhance SR 4233 reduction also caused a marked increase in hepatocyte toxicity and an SR 4233 radical was detected with ESR spectroscopy. SR 4233 cellular reduction and toxicity was prevented with fructose or inhibitors of NADPH:cytochrome P-450 reductase. Inactivation of catalase or glutathione reductase had no effect on SR 4233 toxicity and hepatocyte GSH was not oxidised indicating oxidative stress did not occur during hypoxic SR 4233 hepatocyte toxicity. (3) The lack of SR 4233 cytotoxicity under aerobic conditions could probably be attributed to the detoxification of the SR 4233 radical by mitochondrial oxidation as SR 4233, but not its metabolite SR 4317 markedly increased state III and IV mitochondrial respiration in the presence of NADH. The increased respiration was inhibited by the respiratory inhibitors KCN and antimycin A but not by rotenone. Furthermore SR 4233 cytotoxicity under aerobic conditions was markedly increased by partially inhibiting hepatocytes respiration with cyanide but not rotenone.     

Differential cytotoxicity of diaziquone toward Chinese hamster ovary cells under hypoxic and aerobic exposure conditions

Diaziquone [AZQ, 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone] has been investigated for its toxicity toward Chinese hamster ovary cells AA8-4 under both aerobic and hypoxic conditions. Under acute (1-5 h) exposures to 2.5-10 microM AZQ in alpha-medium plus 10% fetal calf serum, AZQ showed an approximately linear concentration x time dependency for cell killing which was 3-4 times less under hypoxic compared to aerobic conditions. This selective toxicity toward hypoxic cells was prevented by low levels of oxygen. Under aerobic exposure conditions the toxicity of 2.5 microM AZQ was greatly increased by addition of 1-2 mM ascorbate. This ascorbate mediated toxicity of AZQ, presumably extracellular, could be prevented by the simultaneous addition of catalase. Under hypoxic exposure conditions there was no enhancement of AZQ toxicity by ascorbate or protection by catalase. The present results are consistent with two mechanisms for AZQ toxicity proposed earlier by others: toxicity due to (a) redox cycling and increased levels of oxidative stress and (b) reduction of the quinone leading to enhanced reactivity of the aziridines. The relative potency of AZQ as a hypoxic or aerobic cell selective toxin is determined by the balance between these two mechanisms.     

DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9.

Aerobic sensitivity to indoloquinone EO9 has been shown to correlate with cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-electron reducing enzymes in the hypoxic cytotoxicity of EO9. We have characterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines were then selected for a comparison of aerobic and hypoxic sensitivities. Activities of DT-diaphorase showed a wide range (> 10,000-fold), while activities of the one-electron reducing cytochrome b5 and cytochrome P450 reductases were generally lower and showed only a 15- and 25-fold range respectively. The aerobic cytotoxicity of EO9 was clearly related to the cellular levels of DT-diaphorase (r = 0.87), with higher levels giving increased sensitivity, but not to the levels of one-electron reducing enzymes. In contrast, there was no relationship between sensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirapazamine) and activities of any of these reducing enzymes. Under hypoxic conditions sensitivity to EO9 was markedly increased in cell lines with low levels of DT-diaphorase activity, while cell lines with high levels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we have now for the first time demonstrated an inverse correlation (r = 0.93) between the cellular activity of DT-diaphorase and hypoxic sensitivity to EO9, that is sensitivity decreases with increasing DT-diaphorase activity. Moreover, this correlation was lost when cells were exposed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the previously straightforward role for DT-diaphorase in the metabolic activation of EO9. Whereas DT-diaphorase is associated with increased toxicity in air, it appears to reduce the cytotoxicity of EO9 in hypoxic conditions. This suggests either that the one-electron reduction product of EO9 metabolism, the semiquinone, is more toxic than the two-electron reduction product, the hydroquinone, or that the hydroquinone is not cytotoxic and aerobic toxicity is due to the transient appearance of the semiquinone upon back oxidation of the hydroquinone.     

Ascorbate anion potentiates cytotoxicity of nitro-aromatic compounds under hypoxic and anoxic conditions.

The nitro-aromatic radiosensitizing drugs are selectively toxic to hypoxic mammalian cells, and this toxicity can be greatly increased by the addition of ascorbate. The ascorbate itself is not toxic to either hypoxic or aerobic cells (as long as catalase is present to prevent the formation of significant concentrations of hydrogen peroxide) and the mixture of ascorbate plus radiosensitizer is not more toxic to aerobic cells. Sulphydryl reducing agents and dithionite have an effect opposite to ascorbate and decrease the toxicity of nitro-aromatic drugs under hypoxic conditions. Sulphydryl reducing agents are also reported to nullify the radiosensitizing properties of nitro-aromatic drugs, in contrast to ascorbate which has no effect on the radiosensitizing properties. The toxicity of nitro-aromatic drugs decreases rapidly with increasing O2 concentration. This decrease is much less rapid when ascorbate is present. The role of ascorbate in this case may be primarily as an O2 scavenger, although it is also possible that the toxic species produced by radiosensitizer-ascorbate mixtures is less easily removed or detoxified by O2.     

Metabolism of acrylonitrile by isolated rat hepatocytes

Several of the pathways of metabolism of the suspected carcinogen acrylonitrile (AN) were identified previously in this laboratory with the use of subcellular fractions and purified enzymes (Guengerich, F.P., Geiger, L.E., Hogy, L.L., and Wright, P.L., Cancer Res., 41: 4925-4933, 1981). In order to establish the relative contributions of the various pathways leading to activated and detoxicated products, we examined AN metabolism in isolated Fischer 344 rat hepatocytes as a model. Reduced glutathione (GSH) was depleted, and cell viability was lost in an AN concentration-dependent manner. The major GSH adduct formed at all AN concentrations was identified as S-(2-cyanoethyl)GSH using thin-layer and high-performance liquid chromatography. Acid hydrolysis and amino acid analysis of labeled hepatocellular, protein revealed S-(2-carboxyethyl)-cysteine as the major adduct formed, indicating direct alkylation of cysteinyl residues by AN. 2-Cyanoethylene oxide accumulated in the hepatocyte incubations but did not appear to contribute extensively to alkylation of GSH or protein. Cyanide, resulting from hydrolysis of 2-cyanoethylene oxide, appeared to be completely converted to thiocyanate, which was identified by gel exclusion chromatography and mass spectrometry of the methyl derivative. The concentration of thiocyanate formed was directly proportional to the concentration of AN used. Cyanide does not appear to play a role in AN-mediated cell death. Alkylation of hepatocellular DNA and RNA and extracellular DNA was not observed to an extent greater than one base in 3.5 X 10(5). The relative rates of the various pathways were compared, and more than 97% of the metabolites can be accounted for by the described reactions. These results are of use in evaluating the contribution of the various pathways and modes of binding of AN to toxicity and carcinogenicity in liver and extrahepatic target tissues.     

Lymphocytic infiltration and cytotoxicity under hypoxic conditions in the EMT6 mouse mammary tumor

Infiltration of lymphocytes, neutrophils and macrophages was evaluated in hypoxic and well-oxygenated areas of the EMT6 mouse mammary adenocarcinoma, by in vivo staining with the fluorescent dye Hoechst 33342 followed by cell sorting on the basis of fluorescence intensity. Tumors were grouped by days post-injection (days 11-14, 15-17 and 20-27). As lymphocytes are the only host cell population in this tumor model to possess lytic activity against EMT6 tumor cells, the ability of sensitized T lymphocytes to lyse syngeneic EMT6 cells was examined under conditions of varying oxygen concentrations. Infiltrating lymphocytes were detected to the same extent in cell fractions from both areas in all tumors. In contrast, neutrophils were found in significantly higher percentages in the hypoxic population than in the well-oxygenated cell fraction of all but the largest tumors. Macrophages were present in significantly higher percentages in the well-oxygenated fraction than in the hypoxic fraction of day-11 to -14 tumors. Extreme radiobiological hypoxia (0% O2) resulted in a significant decrease in T-cell-mediated lysis of EMT6 tumor cells, compared to lysis in room air (20% O2), but lysis was not impaired under conditions of mild radiobiological hypoxia (1% O2). Our study indicates that host-cell infiltration into areas of differing oxygenation may be quantitated via in situ Hoechst staining followed by cell sorting; in the EMT6 tumor, lymphocytes appear to infiltrate hypoxic areas to the same extent as well-oxygenated areas, and T-lymphocyte killing of syngeneic tumor cells is significantly reduced, although still present, under these hypoxic conditions.     

Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.     

Mechanisms of hypoxic and aerobic cytotoxicity of mitomycin C in Chinese hamster V79 cells.

Mitomycin C (MMC) induced aerobic and hypoxic cytotoxicity in Chinese hamster V79 cells was studied to evaluate the role of the 1-electron versus 2-electron reductive bioactivation. Superoxide dismutase, catalase, and desferal had no protective effects on the aerobic or hypoxic cytotoxicity of MMC, whereas Tempol and Tempol-H, which are known to interrupt and terminate radical reactions, provided partial protection under aerobic conditions. However, under hypoxic conditions, Tempol provided complete protection whereas Tempol-H was ineffective. Electron paramagnetic resonance and spin-trapping investigations, designed to study the mechanisms of such protective effects, confirmed that MMC is activated by the human NADPH:cytochrome P-450 oxidoreductase to its semiquinone radical and that, under aerobic conditions, the semiquinone radical reduces molecular oxygen. Under hypoxic conditions, the semiquinone of MMC reduces H2O2 to produce OH radicals as detected by electron paramagnetic resonance-spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The 1-electron reduced product of MMC was also found to reduce Tempol to the hydroxylamine, Tempol-H, whereas oxidation of Tempol-H by MMC-. was negligible. Cell survival studies and electron paramagnetic resonance observations indicate that the hypoxic cytotoxicity of MMC is mediated by 1-electron activation to its semiquinone intermediate. Under aerobic conditions, the steady state concentration of this intermediate is low due to the facile autooxidation of the semiquinone producing O2-. and H2O2 which are capable of causing oxidative cytotoxicity. Tempol, which can accept an electron from reducing radical species, completely inhibited the hypoxic cytotoxicity of MMC indicating MMC-., the semiquinone of MMC as the species responsible for DNA alkylation and selective hypoxic cytotoxicity of MMC. Our results also indicate that the aerobic cytotoxicity is mediated by other processes in addition to the 1-electron mediated activation.     

Modulation of cisplatin cytotoxicity by sulphasalazine.

The efficacy of cisplatin [cis-diamminedichloroplatinum (II); DDP] is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance (IC50 values of 25.0 +/- 3.9 vs 4.5 +/- 1.0 microM respectively). Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.     

Pharmacokinetics and cytotoxicity of RSU-1069 in subcutaneous 9L tumours under oxic and hypoxic conditions

The acute toxicity, pharmacokinetics and hypoxic cytotoxicity of RSU-1069 were investigated using the subcutaneous (sc) rat 9L tumour model. The pharmacokinetics were studied after i.p. injection of RSU-1069 (20 mg kg-1 or 100 mg kg-1). For both doses, the elimination of RSU-1069 followed first-order kinetics in both plasma and unclamped tumours. After 100 mg kg-1, the peak plasma concentration of RSU-1069 was 40 micrograms ml-1; the elimination t1/2 was 39.3 +/- 11.1 min. After 20 mg kg-1, the peak plasma concentration was 3 micrograms ml-1; the elimination t1/2 was 47.8 +/- 6.3 min. In unclamped tumours, the peak concentration was 50 micrograms g-1 with an elimination t1/2 of 36.1 +/- 9.6 min for the 100 mg kg-1 dose, and 4 micrograms g-1 with an elimination t1/2 of 41.9 +/- 6.1 min for the 20 mg kg-1 dose. The tumour and plasma elimination half-times were not significantly different (P greater than 0.2) for the two doses. Clamping the tumour 30 min after administration of 100 mg kg-1 of RSU-1069 decreased the tumour elimination t1/2 to 10.9 +/- 1.4 min. After releasing the clamp, RSU-1069 returned rapidly to the unclamped tumour concentration. The unclamped tumour/plasma ratio reached a maximum of 4-6, then decreased to a constant value of about 2 for both doses, indicating that RSU-1069 accumulates in these 9L tumours. RSU-1069 kills hypoxic sc 9L cells more efficiently than oxic sc 9L cells; at a surviving fraction of 0.5, the SER was 4.8. For in vitro 9L cells, the SER was approximately 50 when the comparison was between those treated in 2.1% 0(2) and those treated in less than 7.5 x 10(-3)% 0(2); it was approximately 100 when the comparison was between those treated in 21% 0(2) and those treated in less than 7.5 x 10(-3)% 0(3). Tumours treated with RSU-1069 and clamped for various times exhibited biphasic cell-kill kinetics; at 50 mg kg-1, little additional cell kill was achieved after 40 min of clamping. Our data also indicate that RSU-1069 is 300-1000 fold more efficient than misonidazole or SR2508 for killing hypoxic sc 9L tumour cells in situ.     

Active proliferation of mouse hepatocytes in primary culture under defined conditions as compared to rat hepatocytes

Mouse and rat hepatocytes were investigated for replicative potential in primary culture under defined conditions. Mouse hepatocytes replicated at least twice or three times by the 7th day of culture in response to EGF, while only a half of rat hepatocytes replicated once. Thus a far greater potential for proliferation was demonstrated by mouse than rat cells, although the mouse cells ceased dividing by the 10th day of culture. Immunocytochemical examination revealed that the cells produced both albumin and alpha-fetoprotein. Ultrastructural investigation revealed that these cells were immature hepatocytes in nature, based on the presence of microbodies with nucleoids. Thus, primary cultures of mouse hepatocytes seem to be a useful system for studying proliferation and transformation of hepatocytes in vitro.     

Molecular mechanisms of trenimon-induced cytotoxicity in resistant L5178Y/HBM10 cells.

L5178Y/HBM10 lymphoblasts, resistant to a model quinone antitumor agent, hydrolyzed benzoquinone mustard, were approximately 2-fold more sensitive to trenimon (2,3,5-tris-ethyleneimino-1,4-benzoquinone) compared to parental cells (L5178Y). The L5178Y/HBM10 cells are reported to have a 24-fold increased level of DT-diaphorase activity over the parental cells. Inhibition of DT-diaphorase by dicoumarol markedly inhibited the cytotoxic activity of trenimon to the resistant L5178Y/HBM10 cells. Spectrophotometric analysis of the reduction of the quinone, trenimon, to its hydroquinone form was shown to occur approximately 25 times more rapidly in the L5178Y/HBM10 cells relative to the parental cells and was inhibited by discoumarol. Trenimon also induced continuous cyanide-resistant respiration in the L5178Y cells, but not in the resistant L5178Y/HBM10 cells. This suggested a one-electron reduction of trenimon to a semiquinone free radical which could then redox cycle with oxygen in the L5178Y cells. However, in the presence of dicoumarol the resistant L5178Y/HBM10 cells induced similar oxygen activation to the parental cells. Dicoumarol had no effect on trenimon-induced cyanide resistant respiration in the parental cells. These findings suggest that the two-electron reduction of trenimon to its hydroquinone derivative plays a major role in the cytotoxic activity of trenimon.     

Metabolism of N-nitrosodi-n-propylamine and N-nitrosodiallylamine by isolated rat hepatocytes

Isolated rat hepatocytes were incubated with approximately equimolar amounts of N-nitrosodi-n-propylamine (NDPA) and N-nitrosodiallylamine (NDAA) in order to compare their metabolism. The principal metabolite of NDPA was N-nitroso-(2-hydroxypropyl)propylamine, which was present as a glucuronide. N-Nitroso-(3-hydroxypropyl)propylamine and N-nitrosopropyl-(carboxyethyl)amine were minor metabolites; no N-nitrosomethylpropyl-amine (NMPA) was detected. A single N-nitroso metabolite of NDAA was found and identified as N-nitroso-(2,3-dihydroxypropyl)allylamine. These data indicate that the allyl group of N-nitrosodiallylamine is readily oxidized by hepatocytes in vitro. It appears unlikely that N-nitrosomethylpropylamine is an intermediate in N-nitrosodi-n-propylamine metabolism.     

Modulation of 5-fluorouracil catabolism in isolated rat hepatocytes with enhancement of 5-fluorouracil glucuronide formation

The catabolism of 5-fluorouracil (FUra), which accounts for 90% of the elimination of this antimetabolite in vivo, has recently been characterized in freshly isolated rat hepatocytes in suspension using a highly specific high-performance liquid chromatographic methodology. The present study evaluates the effect of thymine and uracil, which are thought to be catabolized by the same enzymes as FUra, on the metabolism and transmembrane distribution of FUra in isolated rat hepatocytes. Following simulataneous exposure of cells for 5 min to 30 microM [6-3H]FUra and increasing concentrations of either thymine or uracil, dihydrofluorouracil (FUH2) levels decreased in a concentration-dependent manner, and the concentration determined for 50% inhibition of FUra catabolism was 8.0 +/- 0.3 (S.D.) and 67.8 +/- 15.6 microM for thymine and uracil, respectively. Analysis of intracellular and extracellular 3H from 1 min to 2 hr after simultaneous incubation of the hepatocytes with 30 microM FUra and thymine (or uracil) in a 1:7 molar ratio resulted in a decrease of intracellular and extracellular FUH2 and alpha-fluoro-beta-alanine (FBAL), while alpha-fluoro-beta-ureidopropionic acid (FUPA) was enhanced. Unmetabolized FUra (not detected in the absence of thymine or uracil) was detected intracellularly in the presence of thymine or uracil and was accompanied by the appearance of a novel metabolite, preliminarily identified as a glucuronide of the FUra base which reached intracellular levels of 44 +/- 9.76 and 27.45 +/- 1.35 microM in the presence of thymine or uracil, respectively, within 1 hr. This metabolite, which penetrates the cell membrane only slowly, accounted for approximately 60% of the intracellular 3H in the presence of 300 microM FUra and 2 mM thymine, whereas FUra catabolism was inhibited by more than 99% under these conditions. The formation of FUra anabolites was insignificant in the presence of thymine and uracil, and incorporation of FUra into RNA was not enhanced. The lack of anabolism of FUra in isolated hepatocytes exposed to either high initial concentrations of FUra or high intracellular FUra concentrations resulting from modulation (inhibition) of FUra catabolism is consistent with the clinical observation of minimal hepatotoxicity with FUra, despite exposure of the liver to high blood levels. These studies indicate that thymine is a more potent modulator of FUra catabolism in hepatocytes than is uracil. Further studies are needed to clarify the biological importance of the glucuronide of the base FUra which accumulates intracellularly as the concentration of FUra increases within the hepatocytes.     

The effect of pH on the aerobic and hypoxic cytotoxicity of SR4233 in HT-29 cells.

We have observed that low pH can substantially potentiate the cytotoxic effect of the bioreductive drug SR4233 in aerobic HT-29 human tumour cells. No such potentiation was observed under hypoxic conditions. This pH effect might be relevant both to the therapeutic effectiveness and to the normal tissue toxicity of this new agent.     

Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes.

The metabolism of 2-acetylaminofluorene (AAF) as well as the activation of AAF to covalently bound and mutagenic intermediates were studied in isolated rat hepatocytes. The cell system readily formed oxidized, deacetylated, and conjugated AAF metabolites. Pretreatments of animals with the inducer beta-naphthoflavone led to increases in phenolic and conjugated as well as covalently protein-bound products. Addition of 4-nitrophenol, a substrate for conjugation, increased the levels of free phenols and inhibited the formation of water-soluble metabolites. At the same time, the rates of covalent protein binding were decreased. Formation of 9-hydroxy-2-acetylaminofluorene could also be demonstrated. The pathway leading to this alicyclic hydroxylated AAF metabolite was not induced by prior beta-naphthoflavone treatment, nor was it inhibited by 4-nitrophenol addition. The cells converted AAF as well as aminofluorene and 2,4-diaminoanisole to mutagenic intermediates which were released into the incubation medium. 2-Aminofluorene was considerably more mutagenic than was AAF in this system. Addition of microsomes increased the mutagenicity of AAF, but not that of 2-aminofluorene or 2,4-diaminoanisole, presumably by deacetylation of N-hydroxy-2-acetylaminofluorene to N-hydroxy-2-aminofluorene.     

原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化

目的：探讨琥珀酸G蛋白偶联受体（GPR91）通路与低氧环境心肌细胞损伤之间的关系。方法：体外培养乳鼠原代心肌细胞，建立低氧模型，再利用siRNA干扰技术降低细胞内GPR91的表达，随机分为阴性对照组（CTL）、CTL+siGPR91组、琥珀酸盐组、琥珀酸盐+siGPR91组、低氧CTL组、低氧CTL+siGPR91组、低氧琥珀酸盐组和低氧琥珀酸盐+siGPR91组。10%氧含量下培养5 d后，CCK-8法检测心肌细胞的存活率、原代心肌细胞低氧模型的细胞ATP含量，Western blot法检测心肌损伤相关蛋白钙调素依赖蛋白激酶Ⅱ（CaMK Ⅱ）、B型尿钠肽（BNP）、GPR91以及PI3K/Akt蛋白通路Akt及P-Akt的表达变化。结果：与CTL组比较，低氧环境下可见心肌细胞存活率降低，ATP含量减少，BNP及CaMK Ⅱ表达上调（均为P < 0.05）；siRNA干扰组心肌细胞GPR91的表达水平下调（P < 0.05），心肌细胞存活率上升、ATP含量增多、BNP及CaMK Ⅱ蛋白表达下调、PI3K及下游信号分子Akt磷酸化水平降低（均为P < 0.05）。结论：GPR91可能通过调控PI3K/Akt通路的磷酸化介入心肌缺血损伤的病理过程。