Prevention of DMBA-induced rat mammary carcinomas comparing leuprolide, oophorectomy, and tamoxifen.

Leuprolide, a gonadotropin releasing hormone agonist, is currently being evaluated in a pilot study of premenopausal women for the prevention of breast cancer. However, little data is available regarding the efficacy of leuprolide in experimental animal models of carcinoma when administered prior to the carcinogen. In the present study the capacity of leuprolide to prevent tumor development was evaluated by comparing its pretreatment effects in the DMBA-induced rat mammary carcinoma model to pretreatment with tamoxifen and oophorectomy. Fifty-five day old, female Sprague-Dawley rats were randomly allocated to one of four groups: 1) no treatment; 2) oophorectomy two weeks prior to DMBA; 3) leuprolide, 40 microg/kg/day; and 4) tamoxifen, 10 mg/kg/week. All animals received four 5 mg doses of DMBA for a total dose of 20 mg. Leuprolide and tamoxifen treatments began two weeks prior to DMBA and ended one week after DMBA administration. Animals were assessed weekly to determine palpable tumor onset, number, size, and volume. At the conclusion of the study (16 weeks), autopsies were performed and tumor tissue was collected for confirmation of malignancy. Seventy-eight percent of the untreated rats developed tumors. No tumors developed in the oophorectomy group, while the number of rats with tumors was significantly reduced (p<0.05) with both leuprolide (30%) and tamoxifen (21.9%) compared to controls (77.8%). There were no significant differences in the tumor number for each tumor-bearing rat or in tumor volume between treated and control groups. Using our dosage regimen, 'chemical oophorectomy' with leuprolide was not as effective as surgical oophorectomy in the prevention of chemical carcinogenesis by DMBA but was comparable to the results obtained with tamoxifen.

Mechanisms for metabolism of ethanol to 1-hydroxyethyl radicals in rat liver microsomes.

Experiments have been designed to reevaluate mechanisms for metabolism of ethanol to 1-hydroxyethyl radicals (HER) in rat liver microsomes. The variables tested include addition of azide, catalase, superoxide dismutase, and deferoxamine, or use of phosphate or Tris buffers. The results indicate that several mechanisms of HER formation are possible, depending on the experimental conditions used to study this process. In the presence of phosphate buffer, which has been used extensively in spite of its ability to chelate iron, HER formation is quite sensitive to changes in hydrogen peroxide availability. These results suggest that Fenton-type reactions produced the oxidizing intermediate responsible for conversion of ethanol to a free radical in phosphate buffer. However, in Tris buffer, HER formation was inhibited markedly by addition of superoxide dismutase, whereas catalase or azide had little effect. These data indicate that the apparent mechanism of radical formation may be influenced by the choice of buffer used. HER formation was almost abolished by the combination of superoxide dismutase and deferoxamine in both buffers, suggesting little enzymatic HER formation by the cytochrome P450 enzymes. When changes in HER formation were compared with rates of ethanol oxidation, it was inferred that 25 to 50% of the acetaldehyde formed during microsomal ethanol oxidation under different experimental conditions could arise via the HER intermediate.

Free radical formation in livers of rats treated acutely and chronically with alcohol.

The spin trapping method was used to assess formation of free radical intermediates in vivo before and after acute alcohol administration to rats. Ascorbyl radicals and spin adducts of dietary alcohol or endogenous compounds, such as lipids, were detected with higher frequency in bile from alcohol-fed rats than in corresponding samples from rats fed control diets. When alcohol was given acutely to these animals, the 1-hydroxyethyl radical metabolite of ethanol was also formed at higher rates in livers of rats that had been fed ethanol chronically. Furthermore, formation of lipid radicals was enhanced after acute alcohol administration. These data support the hypothesis that chronic alcohol administration causes development of oxidative conditions in the liver, which subsequently lead to formation of differing types of radicals. Liver microsomes from alcohol-fed rats also metabolized ethanol to the 1-hydroxyethyl radical at higher rates than controls.

Spin trapping studies of alcohol-initiated radicals in rat liver: influence of dietary fat.

We conducted spin trapping experiments to test the effects of acute and chronic alcohol consumption in livers from rats that had been fed either high fat (35% of energy) or low fat (12% of energy) liquid diets. Rats were anesthetized with isoflurane, the spin trapping agent POBN [alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone] was administered by intravenous injection, and bile samples were collected for electron paramagnetic resonance (EPR) analyses. Two different types of EPR spectra were observed in bile from the animals in these studies. One set of spectral lines was from the 1-hydroxyethyl radical adduct of POBN, which was conclusively identified by injecting the rats with [1-13C]ethanol. The EPR signals of a second type of radical adduct in bile could be observed both before and after acute administration of ethanol. Although the radical(s) responsible for this second series of signals could not be conclusively identified, it is likely that lipid radicals were formed under these conditions and trapped by POBN. For both types of radical adducts, the most intense EPR signals were observed in rats that had been fed alcohol in combination with a high fat diet for 2 wk before the experiments. These results confirm and extend previous data indicating that high levels of dietary fat enhance alcohol-associated free radical formation in the liver.

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes.

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.

Degradation of DMPO adducts from hydroxyl and 1-hydroxyethyl radicals by rat liver microsomes.

Hydroxyl and 1-hydroxyethyl radical adducts of 5,5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenan-throlene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe+2 and K3Fe(CN)6, but Fe+3 alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes from superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe+2, and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.

In vivo spin trapping of nitric oxide generated in the small intestine, liver, and kidney during the development of endotoxemia: a time-course study.

Spin trapping of nitric oxide (NO.) in vivo in liver, small intestine, kidney, and plasma of intact rats was accomplished using diethyldithiocarbamate (DETC) administered intraperitoneally. DETC combines with Fe2+ to form (DETC)2-Fe and is an excellent trapping agent for nitric oxide. DETC distribution and uptake by the organs of interest was determined and the formation of the active trapping agent (DETC)2-Fe was assayed in the various organs and plasma. The capacity of this spin trap to capture NO. in vivo was demonstrated by administering sodium nitroprusside to the animals. The trapping procedure was then used to assess the course of NO. generation during a 6 h period in animals that had been treated with endotoxin. The rate of NO. generation/gram tissue was determined during the last 15 min of each time period. The results indicate that induction of nitric oxide generation begins earliest in the small intestine, then in the liver, and still later in the kidney and plasma. Nitric oxide production was most intense in the liver and was still increasing at the end of the experiment. Control animals receiving the spin trapping agent showed only little or no evidence of nitric oxide production except for the small intestine. The results show that induction of NO. generation caused by endotoxin begins at different times in different organs.

Development of circulatory and metabolic shock following transient portal triad occlusion.

Liver ischemia is purposefully induced by portal triad occlusion (PTO) in several clinical situations including liver surgery for trauma, tumor, and transplantation. Despite significant morbidity from PTO, the hemodynamic and metabolic effects of PTO have not been evaluated relative to duration of ischemia. We investigated this using a total hepatic ischemia model. Rats received isoflurane anesthesia, carotid artery and jugular vein cannulation, and serial measurements of cardiac output (CO), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), stroke volume (SV), systemic vascular resistance (SVR), superior mesenteric artery blood flow (SMAF), intestinal vascular resistance (IVR), pH, pCO2, pO2, lactate, glucose, hematocrit (HCT), white blood cell count (WBC), and total neutrophils. Each group received 0, 15, 30, 45, or 60 min of PTO followed by 2 hr of reperfusion. All sham ischemia animals remained hemodynamically stable throughout the study. However, in the ischemic groups, there were significant time-dependent decreases in MAP, HR, CO, CVP, SV, SMAF, and pH, and increases in SVR, IVR, HCT, and lactate, while pCO2, pO2, glucose, and WBC remained stable. All of the ischemic animals survived except those that received 60 min of PTO. In this group, all of the animals survived the ischemic period; however, only one animal survived beyond 60 min of reperfusion. These data demonstrate a time-dependent circulatory and metabolic shock following PTO heralded by intestinal venous pooling and loss of intravascular fluid, and culminating in death. Careful hemodynamic monitoring and restoration of blood volume in the trauma patient may reduce morbidity and mortality.

Recurrent ischemia in the canine heart causes recurrent bursts of free radical production that have a cumulative effect on contractile function. A pathophysiological basis for chronic myocardial "stunning".

Open-chest dogs (total number used, 117) underwent 10 5-min coronary occlusions (O) interspersed with 10 min of reperfusion (R). When systolic thickening fraction was measured 9 min after each R, the first O-R cycle was found to cause the largest decrement, with only a slight additional loss during the next four cycles and no further loss during the last five cycles (group IV), suggesting that the first few episodes of ischemia preconditioned the myocardium against the stunning induced by the last five episodes. However, different results were obtained when the total deficit of wall thickening during the final 4-h R interval was measured. The total deficit was similar after one and three 5-min O (groups V and VI, respectively), indicating that the first ischemic episode did precondition against the next two episodes; however, it was approximately 2.5-fold greater after 10 O (group IV) than after 3, indicating that the first 3 episodes failed to precondition against the next 7. Thus, at some point between the 4th and 10th O, the preconditioning effect was lost and recurrent ischemic episodes started to have a cumulative effect. Measurements of free radicals with alpha-phenyl N-tert-butyl nitrone (PBN) demonstrated a burst of free radical generation immediately after the 1st, 5th, and 10th R (group VIII). The total cumulative release of PBN adducts during the initial 5 min of reflow was 58% less after the 5th R than after the 1st (P < 0.05) but did not differ significantly between the 1st and 10th R. When administered throughout the 10 O-R cycles, the .OH scavenger mercaptopropionyl glycine significantly enhanced the recovery of function (group I) and markedly suppressed the formation of free radicals (group VII). However, the beneficial effects of mercaptopropionyl glycine were completely, or largely, lost if the drug was discontinued after the first five (group II) or eight (group III) O-R cycles, respectively, implying that (a) the oxidative stress associated with the last five, or even two, cycles was sufficient to cause severe postischemic dysfunction, and (b) the cumulative injury caused by repetitive ischemic episodes is mediated by recurrent oxidative stress. This study provides direct in vivo evidence that oxygen radicals play an important role in the pathogenesis of myocardial stunning after repetitive ischemia, and implicates .OH as a primary culprit. Taken together, the data indicate that recurrent brief ischemic episodes result in recurrent bouts of oxyradical-mediated injury that have a cumulative effect on contractility, a situation that could lead to protracted or even chronic myocardial stunning.

Metabolism of ethanol to 1-hydroxyethyl radicals in vivo: detection with intravenous administration of alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone.

Intravenous administration of the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) to anesthetized but otherwise untreated rats was used to test for formation of 1-hydroxyethyl radicals in vivo. The only EPR signals observed in bile samples from rats that had received ethanol but no POBN could be attributed to low concentrations of ascorbyl radical. However, when POBN (700 mg/kg, intravenously) was also administered, a nitroxide with a six-line EPR spectrum was readily detected in bile. This spin adduct was proven to be the 1-hydroxyethyl radical adduct of POBN through injection of [1-13C]ethanol to rats, which resulted in the presence of an adduct with a 12-line EPR spectrum. Comparable results were obtained in experiments with isolated perfused rat livers. 1-Hydroxyethyl radical spin adducts of POBN were readily detectable in bile in the presence of only moderate (10-15 mM) concentrations of alcohol. In these experiments, bile samples were collected into a mixture of dipyridyl and bathocuproine disulfonic acid, and the effectiveness of these chelators to prevent ex vivo signal formation was confirmed experimentally. No EPR signals for nitroxide spin adducts were observed in plasma or perfusate, even though high concentrations of POBN and alcohol were present. Taken together, these data indicate that 1-hydroxyethyl radicals are formed in vivo and can be readily detected in bile when high concentrations of POBN are achieved through intravenous injection.

A new model for inducing total hepatic ischemia while preventing circulatory collapse secondary to splanchnic vascular congestion.

Animal models used to study liver ischemia are limited by the lethal effect of splanchnic venous engorgement from portal triad occlusion (PTO). We compared a passive porto-systemic shunt (PSS) to a pump-driven PSS. The passive and pumped PSS groups (n = 6) received 60 min of PTO followed by 2 h of reperfusion. A control group received all interventions, but no PTO, and remained stable throughout. During PTO, severe circulatory shock with intestinal ischemia occurred in the passive group, while the pumped group remained stable. During reperfusion, both shunted groups experienced varying degrees of metabolic acidosis with decreases in cardiac index, stroke volume, superior mesenteric artery flow, and increases in systemic and intestinal vascular resistance. The mortality rate for the passive group was 83% vs. 0% for the pumped group. These results suggest that pumped PSS prevents splanchnic engorgement and allows for reproducible, isolated total hepatic ischemia in vivo.

Inhibitory effect of a hydrophilic alpha-tocopherol analogue, MDL 74,405, on generation of free radicals in stunned myocardium in dogs.

A previous study has demonstrated that the hydrophilic (alpha-tocopherol analogue, MDL 74,405, attenuates postischemic myocardial dysfunction ("stunning") in dogs. The present study was undertaken to determine directly whether the salutary effect of this drug on myocardial stunning results from inhibition of the generation of oxygen-derived free radicals. Open-chest dogs undergoing a 15-min coronary artery occlusion and 3 h of reperfusion received an intravenous infusion of either saline (controls, n = 7) or MDL 74,405 (n = 6) starting 30 min before coronary occlusion and ending 60 min after reflow at a dose of 0.3 mg/kg/h. To measure free radical production, all dogs received an intravenous infusion of the spin trap alpha-phenyl N-tert-butyl nitrone (PBN) and local coronary venous plasma was analyzed by electron paramagnetic resonance (EPR). In control dogs, the myocardial production of PBN adducts exhibited an initial burst immediately after the onset of reflow and remained elevated until 10 min after reperfusion. Dogs treated with MDL 74,405 demonstrated a marked decrease in PBN adduct production. This effect of MDL 74,405 could not be attributed to nonspecific factors such as differences in ischemic zone size, collateral flow, arterial pressure, heart rate, coronary flow or other hemodynamic variables. These results demonstrate that the hydrophilic vitamin E analogue, MDL 74,405, inhibits free radical generation after myocardial ischemia-reperfusion in vivo. This finding provides direct evidence that the salutary effects of MDL 74,405 on myocardial stunning are due to attenuation of oxidative stress.

Inorganic phosphate promotes redox cycling of iron in liver microsomes: effects on free radical reactions.

The phosphate buffer concentration used in spin trapping experiments with liver microsomes markedly influenced rates of free radical formation from ethanol and dimethylsulfoxide, but not from carbon tetrachloride. Effects of phosphate concentration on ethanol radical formation were abolished by addition of deferoxamine or bathophenanthrolene, indicating that an iron-phosphate complex might be involved. High concentrations of phosphate stimulated rates of microsomal Fe+3 reduction and facilitated the mobilization of microsomal nonheme iron, but had little effect on a variety of microsomal monooxygenase enzyme activities. Although microsomal oxygen utilization and superoxide production were relatively unaffected by phosphate, hydrogen peroxide concentrations were markedly decreased in the presence of high concentrations of phosphate. Taken together, the data suggest that a ferric-phosphate complex may be enzymatically reduced by microsomal enzymes and NADPH. Reoxidation of ferrous ion is nonenzymatically promoted by phosphate and/or H2O2 produced by the microsomes. During the process of reoxidation, one or more oxidizing intermediates may be formed which initiate secondary free radical reactions. Although the reactivity of the intermediate(s) is similar to that of the hydroxyl radical, no spin trapping evidence was obtained to support this assignment.

Inhibition of doxorubicin-induced membrane damage by thiol compounds: toxicologic implications of a glutathione-dependent microsomal factor.

The hypothesis was tested that glutathione exerts its protective actions against doxorubicin-induced oxidative stress through an enzyme-dependent mechanism. Glutathione at biological concentrations decreased doxorubicin-dependent rat hepatic microsomal lipid peroxidation, whereas N-acetylcysteine had no effect. Glutathione was utilized during this inhibition at a rate dependent on the concentration of both doxorubicin and the sulfhydryl. Increasing glutathione concentrations resulted in significant increases in utilization. N-acetylcysteine was also oxidized in the microsomal system; however, the rate of oxidation was not enhanced by doxorubicin. If bovine cardiac microsomes were substituted for the hepatic microsomes, no lipid peroxidation was detected in the presence of doxorubicin, yet significant utilization of glutathione was detected. Microsomes isolated from tocopherol-deficient rats utilized less glutathione in the presence of doxorubicin, and there was no inhibition of doxorubicin-dependent lipid peroxidation. These findings support the conclusion that glutathione inhibits hepatic microsomal lipid peroxidation initiated by the redox-cycling of doxorubicin. Inhibition of doxorubicin-dependent lipid peroxidation appears to be enzyme-mediated and to require tocopherol. A similar mechanism for protection against doxorubicin appears to be present in heart microsomal membranes.

Study of reproducibility of spin trapping results in the use of C-phenyl-N-tert-butyl nitrone (PBN) for trichloromethyl radical detection in CCl4 metabolism by rat liver microsomal dispersions. Biological spin trapping I.

The well-known metabolism of CCl4 to trichloromethyl radicals in rat liver microsomal dispersions has been reinvestigated with the goal to determine the repeatability and reproducibility of the EPR signal intensity of the EPR spectrum of the CCl3 adduct of PBN. It was found that at least eight repeat experiments were needed under identical conditions to obtain an average value with an error of +/- 10%. When the effect of changing the concentrations of CCl4, PBN or NADPH-generating system was investigated, the plots of EPR signal intensity vs. the variable selected showed initial smooth increases in signal strength with respect to an increase in concentrations of CCl4, PBN or NADPH-generating system. However, considerable scatter was found after the initial slope and only general trends could be recognized. It is concluded that with CCl4, no increase in EPR signal is found after 10 mM concentration. For PBN, the optimum concentration is about 30 mM. The signal strength seems to increase with increased amounts of NADPH generating system although with diminishing slope.

Catechol adrenergic agents enhance hydroxyl radical generation in xanthine oxidase systems containing ferritin: implications for ischemia/reperfusion.

Iron chelators have been reported to protect tissues against reperfusion injury. This implies that iron is being released into the plasma or is made accessible in tissues for oxidation-reduction reactions. It has been postulated that ferritin is a likely source for this iron. This report demonstrates that adrenergic agents with the catechol structure, which includes the endogenous catecholamines norepinephrine and epinephrine, are capable of releasing iron from ferritin. It is shown that the net release of iron from ferritin by epinephrine is significantly enhanced under anaerobic conditions. The findings suggest that catecholamines can mediate iron release from ferritin under conditions that can occur during ischemia/reperfusion. Catecholamines are also shown to interact with the released iron and xanthine oxidase to produce highly reactive hydroxyl radicals. The implications of this interaction for ischemia/reperfusion are discussed.

Metabolism of ethanol to 1-hydroxyethyl radicals in rat liver microsomes: comparative studies with three spin trapping agents.

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl3, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.

Characteristics of an oxidant formed during iron (II) autoxidation.

When ethanol (100 mM) and the spin trapping agent DMPO were added to a solution of FeSO4 (0.1 mM) in phosphate buffer (40 mM), pH 7.4, the spectrum of the 1-hydroxyethyl radical spin adduct of DMPO could be detected by ESR spectroscopy. The ESR signal intensity of the 1-hydroxyethyl radical increased with higher concentrations of phosphate. Under these conditions, hydroxyl radical adducts could not be detected. However, if H2O2 (0.1 mM) was also added, the ESR spectra contained signals from both the hydroxyl and 1-hydroxyethyl radical adducts of DMPO. When ethanol was replaced with azide (100 mM) in these experiments, strong ESR signals from the azidyl radical adduct of DMPO were observed only in the Fenton system. The absence of hydroxyl radical adduct signals in the Fe(2+)-PO4 reaction could not be explained by instability of the hydroxyl radical adduct in the presence of Fe2+ or superoxide. Experiments with oxygen radical scavengers indicated that the oxidant formed during Fe2+ autoxidation was less reactive to benzoate, DMSO and tert-butanol than the hydroxyl radical. The available data indicate that the primary oxidant formed during Fe2+ autoxidation in phosphate buffer is not the hydroxyl radical, but may be an iron-oxygen complex such as a ferryl species.

Oxygen radical formation in well-washed rat liver microsomes: spin trapping studies.

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5,5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O2, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1,2,4-triazole in the presence of H2O2 were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.