Xenoestrogens significantly enhance risk for breast cancer during growth and adolescence.

Breast cancer is one of the most common forms of cancer observed in women, and endogenous estrogen is thought to play a major role in its development. Because of this, any conditions or exposures which enhance estrogenic responses would result in an increased risk for breast cancer. The role of xenoestrogenic compounds, such as DDT, in the etiology of breast cancer is still very controversial. In the following paper we discuss recently-published observations by ourselves and others which indicate that xenoestrogens may play a significant role in the development of breast cancer. Specifically, we hypothesize that during periods of high growth rates and during breast development the sensitivity of breast cells to estrogenic compounds is sufficiently great for xenoestrogens to significantly enhance risk for breast cancer.

Running exercise may reduce risk for lung and liver cancer by inducing activity of antioxidant and phase II enzymes.

The effects of exercise, ethanol, and exercise plus ethanol-treatments on activity of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UDP-GT) in lung and liver were investigated. All treatments induced SOD and CAT activity in the lung while CAT activity was enhanced only by the combined treatments in the liver. Ethanol reduced hepatic SOD activity, while with the combined treatment SOD was normal. Exercise enhanced UDP-GT activity in liver and lung while ethanol had no effect and GST activity was induced in the liver by the combined treatment. Thus exercise may reduce risk for lung and hepatic cancer and prevent an ethanol-induced increase in risk for hepatic cancer by enhancing activity of antioxidant and phase II enzymes.

Estrogenic and DNA-damaging activity of Red No. 3 in human breast cancer cells.

Exposure to pesticides, dyes, and pollutants that mimic the growth promoting effects of estrogen may cause breast cancer. The pesticide DDT and the food colorant Red No. 3 were found to increase the growth of HTB 133 but not estrogen receptor (ER) negative human breast cells (HTB 125) or rat liver epithelial cells (RLE). Red No. 3, beta-estradiol, and DDT increase ER site-specific DNA binding to the estrogen response element in HTB 133 cells and increase cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells. Site-specific DNA binding by p53 in RLE, HTB 125, HTB 133, and MCF-7 cells was increased when they were treated with Red No. 3, which suggests that cellular DNA was damaged by this colorant. Red No. 3 increased binding of the ER from MCF-7 cells to the estrogen-responsive element. Consumption of Red No. 3, which has estrogenlike growth stimulatory properties and may be genotoxic, could be a significant risk factor in human breast carcinogenesis.

Induction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activation in rat lung microsomes by chronic ethanol consumption and repeated running exercise.

The effects of both chronic ethanol consumption and repeated exercise on metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by rat lung microsomes were investigated. Keto aldehyde production was significantly enhanced 52% and 98%, respectively, by the ethanol and exercise, as was keto alcohol production (72% and 76%). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) production was significantly enhanced only by the exercise (+32%). The combined treatment enhanced the keto alcohol production by 39%, indicating non-additive effects on cytochrome P450 (CYP) isozymes by the different treatments. Immunoblot and metabolic studies revealed an increased activity and content of CYP1A2 and CYP2B induced by the ethanol and exercise treatments as well as an induction of CYP2E1 proteins by the ethanol and combined treatments. These results indicate that both ethanol consumption and running exercise enhance NNK activation by increasing the expression of the CYP enzymes responsible for NNK activation.

Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.

Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.

Induction of HSP72 in rat liver by chronic ethanol consumption combined with exercise: association with the prevention of ethanol-induced fatty liver by exercise.

Ethanol-induced fatty liver in rats was attenuated by repeated running exercise, and the protective effect of exercise was associated with the synergistic expression of heat shock proteins (HSP72). Rats were placed in four groups of six. The two ethanol-fed groups of rats received a liquid diet (Lieber-DeCarli formulation) in which 36% of the calories were derived from ethanol. One group remained sedentary (S/E), whereas the other was trained to run on a rodent treadmill at a speed of 27 m/min, 1 hr/day, 5 days/week, for 7 weeks (R/E). Two other groups--one exercised as previously mentioned (R/C) and one sedentary (S/C)--received control-liquid diets in which the ethanol was isocalorically substituted with a dextran/maltose mixture. The degree of fatty infiltration in liver sections stained with hematoxylin and eosin was graded on a 0-4 scale and the data analyzed by ANOVA on ranks. Ethanol significantly induced fatty infiltration in the S/E group, whereas fatty infiltration in the livers of the R/E group was not different from the S/C group. Electrophoresis and Western blotting of liver homogenates demonstrated that HSP72 was not expressed in either the S/C or S/E groups and was only slightly expressed in the R/C group. The combination of exercise and ethanol, however, resulted in an elevated expression of HSP72 in the R/E group. The content of HSP73 was unaffected by any treatment.

Effect of swimming exercise and ethanol on rat liver P450-dependent monooxygenases.

The interactive effects of 6 wk of repeated swimming exercise and chronic ethanol consumption (36% of total calories) on the hepatic cytochrome P450-dependent monooxygenase system were studied utilizing four groups of male rats in a 2 x 2 factorial design. The sedentary-control (S/C), sedentary-ethanol (S/E), and swim-control (SW/C) groups received the same amount of food that the swim-ethanol (SW/E) group consumed. The swimming groups were trained to swim for 2 h.d-1, 5 d.wk-1. Significant main effects due to ethanol (P < 0.002) and exercise (P < 0.02) were observed for the enhanced cytochrome P450 content and cytochrome P450 reductase activity, respectively. In addition, significant main effects for ethanol (P < 0.001), exercise (P < 0.0001), and significant interaction effects (P < 0.005) on aniline p-hydroxylase activity and significant main effects for ethanol (P < 0.01), exercise (P < 0.01), and interaction effects (P < 0.04) on 7-ethoxycoumarin o-deethylase activity were observed. Because the SW/C treatment had no effect on any of the measured cytochrome P450 activities and the SW/E treatment enhanced P450 activities much more than the S/E treatment, the main effects observed for exercise are accounted for by the alterations produced by combining swimming with the ethanol treatment. Based on these results, repeated exercise combined with ethanol consumption produces a synergistic increase in ethanol-inducible cytochrome P450-dependent activities.

Antibodies raised against trifluoroacetyl-protein adducts bind to N-trifluoroacetyl-phosphatidylethanolamine in hexagonal phase phospholipid micelles.

The delayed fulminant form of halothane hepatotoxicity is thought to be triggered by an immune response to haptenic adducts formed by a metabolite, trifluoroacetyl chloride. In this study we demonstrate that antibodies purified from the sera of rabbits sensitized to a trifluoroacetyl-protein adduct will cross-react with a trifluoroacetyl-phosphatidylethanolamine adduct. Trifluoroacetyl adducts of both rabbit serum albumin (TFA-RSA) and dioleoylphosphatidylethanolamine (TFA-DOPE) were prepared. The TFA-RSA was coupled to an Affigel-10 affinity column to purify hapten-selective immunoglobulin (Ig) G antibodies (anti-TFA-RSA IgG) from the sera of rabbits given i.m. injections of TFA-RSA. The TFA-DOPE was purified by high-performance liquid chromatography and the structure confirmed with direct chemical ionization mass spectrometry. Lamellar liposomes containing a mixture of 5% TFA-DOPE, 71% DOPE and 24% dioleoyl-phosphatidylcholine, as well as hexagonal phase micelles containing 5% TFA-DOPE and 95% DOPE, were prepared by sonication. Anti-TFA-RSA IgG antibodies were added to each of these lipid mixtures for 30 min, fluorescein-conjugated goat-antirabbit IgG antibodies were added next for an additional 30 min and then binding of anti-TFA-RSA IgG antibodies to TFA-DOPE was quantified by flow cytometry. Anti-TFA-RSA IgG antibodies bound to TFA-DOPE only when it was incorporated into hexagonal phase micelles. These findings suggest that TFA-phosphatidylethanolamine adducts that reside in nonlamellar domains on the hepatocyte surface could be recognition sites for anti-TFA-adduct antibodies and potentially participate in immune-mediated hepatotoxicity.

Binding of anti-acetaldehyde IgG antibodies to hepatocytes with an acetaldehyde-phosphatidylethanolamine adduct on their surface.

We have previously shown that antibodies raised against acetaldehyde adducts of protein cross-react with an acetaldehyde adduct of dioleoylphosphatidylethanolamine, N-ethyl-dioleoylphosphatidylethanolamine, when the latter is incorporated into hexagonal phase phospholipid micelles. In the present study we demonstrate that these same IgG antibodies cross-react with N-ethyl-dioleoylphosphatidylethanolamine when this adduct is incorporated into the surface of hepatocytes. Hapten-specific IgG antibodies were purified from the sera of rabbits sensitized to an albumin-acetaldehyde conjugate that had been reduced with sodium cyanoborohydride (N-ethyl-RSA). The N-ethyl-RSA was coupled to an Affi-Gel-10 column to affinity purify the IgG. Liposomes containing N-ethyl-dioleoylphosphatidylethanolamine were fused with isolated hepatocytes, the affinity purified primary IgG antibodies were added, then fluorescein-conjugated second antibodies were added, and antibody binding to hepatocytes was measured by flow cytometry. The fluorescence of these hepatocytes was significantly greater (p less than 0.01) than control hepatocytes prepared with (1) pre-immune primary IgG antibodies with fluorescein-conjugated second antibodies, (2) no primary antibody but with fluorescein-conjugated second antibodies, and (3) no fluorescein-conjugated second antibodies.

Cross-reactivity of antibodies raised against acetaldehyde adducts of protein with acetaldehyde adducts of phosphatidyl-ethanolamine: possible role in alcoholic cirrhosis.

This study measured the possible cross-reactivity of hapten-specific IgG antibodies purified from the sera of rabbits sensitized to an albumin-acetaldehyde conjugate [N-ethyl-rabbit serum albumin (N-ethyl-RSA)] with acetaldehyde-phosphatidylethanolamine adducts. The N-ethyl-RSA was coupled to an Affigel-10 column to affinity purify the IgG (anti-N-ethyl-RSA IgG). Dioleoyl-phosphatidylethanolamine (DOPE) was reacted with acetaldehyde to form a Schiff base, which was reduced to N-ethyl-DOPE, purified by high pressure liquid chromatography, and analyzed with direct chemical ionization mass spectrometry. Lamellar liposomes containing either 5% by weight N-ethyl-DOPE and 95% egg phosphatidylcholine or a mixture of 5% N-ethyl-DOPE, 71% DOPE, and 24% dioleoylphosphatidylcholine, as well as hexagonal phase micelles containing 5% N-ethyl-DOPE and 95% DOPE, were prepared by sonication. Anti-N-ethyl-RSA IgG was then incubated with each of these lipid mixtures for 30 min, a fluorescein-conjugated goat anti-rabbit IgG was added for an additional 30 min, and then binding of anti-N-ethyl-RSA IgG to N-ethyl-DOPE in the liposomes or micelles was measured by flow cytometry. Anti-N-ethyl-RSA IgG bound to N-ethyl-DOPE in both vesicles and hexagonal phase micelles, but the affinity was 16 times greater for the hapten in the hexagonal phase. This result demonstrates that physical presentation of the hapten can affect antibody recognition and that antibodies raised against N-ethyl-RSA can cross-react with acetaldehyde-phospholipid adducts.

Both acute and chronic exercise enhance in vivo ethanol clearance in rats.

Rates of ethanol clearance were measured at rest and with acute exercise in four groups of female Sprague-Dawley rats. Two groups were trained to run on a motor-driven rodent treadmill at 27 m/min, 1 h/day, 5 days/wk and were given a nutritionally balanced liquid diet; one of these groups received 35% calories as ethanol whereas in the other, sucrose was isocalorically substituted for the ethanol. Appropriate sedentary and nonethanol controls were also used. Clearance of a 1.75-g/kg ethanol dose injected intraperitoneally was determined by measuring ethanol levels in the blood each hour and utilizing these values in the Widmark equation (R. Teschke, F. Moreno, and A. Petrides, Biochem. Pharmacol. 30: 1745-1751, 1981) for calculating whole-body ethanol clearance. Rates of ethanol clearance were determined for each rat at 4 and 7 wk of training. The clearance tests at 4 wk included a 60-min period of running exercise, whereas the tests 3 wk later were conducted at rest. The results indicate that both acute exercise and exercise training can increase rates of in vivo ethanol clearance. In addition, the chronic exercise appeared to increase in vitro ethanol metabolism by hepatic microsomes without altering in vitro hepatic alcohol dehydrogenase activity.

Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes. Effects of prior treatment with ethanol and other xenobiotics.

The cytochrome P-450 monooxygenase system of hamster liver microsomes and its response to prior treatment with ethanol and other xenobiotics have been examined. Male Syrian golden hamsters were administered ethanol (ETOH), phenobarbital (PB), 5,6-benzoflavone (BF) or isoniazid (INH). Each treatment resulted in a moderate increase (20-60%) in the specific content of liver microsomal cytochrome P-450 along with a unique hemeprotein ferrous carbonyl Soret maximum. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of liver microsomes revealed distinctive changes in protein banding patterns in the cytochrome P-450 (45-60 kDa) region with each treatment. NADPH: cytochrome c reductase activity was increased by both PB and INH, whereas cytochrome b5 content was increased by INH only. Microsomal oxidation of ETOH and aniline p-hydroxylation (expressed per nmol cytochrome P-450) were enhanced dramatically by ETOH and INH, whereas PB and BF had no effect on these enzymatic activities. Both ETOH and INH also increased zoxazolamine 6-hydroxylation but, in contrast to other rodent species, this drug-metabolizing activity was decreased in hamster liver microsomes after treatment with either PB or BF. Microsomal benzphetamine N-demethylation was decreased by ETOH, INH and BF administration and was only modestly enhanced after treatment with PB. ETOH and INH had no effect on the O-deethylation of 7-ethoxycoumarin, and enzymatic activity increased by BF but decreased by PB. These results demonstrate that the cytochrome P-450-dependent monooxygenase system of hamster liver microsomes responds to treatment with ETOH and other xenobiotics in a manner that is quantitatively and, in certain respects, qualitatively different from that reported for the rat, rabbit, and mouse.

Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography.

NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.

Effects of exercise and ethanol on liver mitochondrial function.

Rates of ADP stimulated respiration for various substrates were determined in mitochondria isolated from the livers of female Sprague-Dawley rats following 8 weeks of treatment with daily swimming, ethanol consumption, or both. All rats were fed an American Institute of Nutrition (AIN) type liquid diet with the ethanol treated rats receiving 35% of the calories as ethanol. Chronic exposure to ethanol depressed both state 3 respiration with glutamate as a substrate and cytochrome oxidase activity. Respiratory control ratios and P:O ratios, however, were unaffected by the ethanol exposure. Exercise alone had no effect on hepatic mitochondrial function. There were also no significant alterations in oxidative function of hepatic mitochondria from rats which were endurance-trained by swimming while receiving the ethanol diet. This lack of alteration in mitochondrial function was in spite of the fact that these rats consumed an identical amount of ethanol as those which incurred mitochondrial dysfunction. These results indicate that regular exercise has the potential to attenuate the ethanol induced decline in hepatic mitochondria.

The interaction of ethanol and swimming upon cardiac mass and mitochondrial function.

Four groups of female Sprague-Dawley rats received a nutritionally adequate liquid diet formulated for rats. Two groups, one ethanol diet and one control diet swam 6 days/wk for 6 weeks and were designated swim ethanol (SWM-E) and swim control (SWM-C) respectively. Their swimming time increased from 15 min/day on the first day to 2 hrs/day during the final week. One sedentary group received an ethanol diet (SED-E) while another sedentary group received a control diet (SED-C). In the ethanol diet 35% of the calories as ethanol isoenergetically replaced dextrin. The group mean body weights were not different at the end of 6 weeks. The left ventricles of both swimming groups showed similar gains in weight, 13% for the ethanol and 15% for the control. Mitochondrial respiration in the ethanol groups showed a significant depression across substrates and across both pupulations of mitochondria (subsarcolemmal and intermyofibrillar). The swimming-ethanol interaction in the SWM-E group caused an atrophy of the gastrocnemius-plantaris muscle as evidenced by the 13% loss in weight of the muscle. We conclude that chronic ingestion of ethanol will suppress mitochondrial respiration in sedentary and swimming exercised rats, but will not suppress cardiac hypertrophy in the swimming exercised rats. Muscles that are not chronically overloaded by swimming, such as the gastrocnemius-plantaris muscles will undergo atrophy during the swimming protocol of 6 weeks.

The interaction of aging and endurance exercise upon the mitochondrial function of skeletal muscle.

Mitochondrial function was determined in sedentary-control animals, 150, 300 and 720 days of age, and in endurance-trained animals 300 and 720 days of age. The mitochondria were isolated from two regions of the cell of the gastrocnemius-plantaris muscle, subsarcolemmal and intermyofibrillar. State 3 respiration did not change with increasing age in control animals, but endurance training enhanced state 3 respiration in both the 300 and 720 day old trained animals. Age decreased the amount of intermyofibrillar mitochondrial protein, while training increased the mitochondrial protein of both regions of the cell. The decrease in oxidative metabolism in the skeletal muscle resulted from a decrease in mitochondrial protein, not to a decrease on mitochondrial function.