Biomedical subjects
Publications and source records attributed to A H Conney.
The administration of green tea, black tea, or (-)-epigallocatechin gallate inhibited the growth of established nonmalignant and malignant tumors in tumor-bearing mice. In experiments with black tea, we found that its oral administration inhibited DNA synthesis and enhanced apoptosis in both nonmalignant and malignant tumors in tumor-bearing mice.
The hypothesis that the decrease in the proportion of mutations at AT base pairs in Chinese hamster V-79 cells treated with increasing doses of (+)-(R,S,S,R)-benzo[a]pyrene diol epoxide ((+)-BPDE) is due to saturation of A for adduct formation was investigated by comparing the ratio of dA to dG adducts formed at high (0.48 microM) and low (0.04 microM) doses of [3H]-labeled (+)-BPDE. The dA to dG adduct ratio was similar in both calf thymus DNA and the genomic DNA in V-79 cells, and did not change with dose. For the V-79 cells, this ratio was also unaffected by a 24-h post treatment repair incubation.
Catechol estrogens (2- or 4-hydroxyestradiol and 2- or 4-hydroxyestrone) are chemically reactive estrogen metabolites that are O-methylated to less polar monomethyl ethers by catechol-O-methyltransferase, an enzyme present in many tissues such as the liver, kidney, brain, placenta, uterus, and mammary gland. In the present report, we review recent studies on the antitumorigenic and antiangiogenic effects of exogenously administered 2-methoxyestradiol in vitro and in vivo. We also discuss data that suggest that endogenous formation of 2-methoxyestradiol (and its 2-hydroxyestradiol precursor) may have a protective effect on estrogen-induced cancers in target organs. Although the molecular mechanism of action of 2-methoxyestradiol is not clear, we suggest that some unique effects of 2-methoxyestradiol may be mediated by a specific intracellular effector or receptor that is refractory to the parent hormone, estradiol. Additional research is needed to identify factors that regulate the metabolic formation and disposition of 2-methoxyestradiol in liver and in target cells and to evaluate the effects of modulating 2-methoxyestradiol formation on estrogen-induced carcinogenesis.
In female rats, total estrone-3-sulfatase activity per liver in the nuclear fraction is comparable to the total activity per liver in the microsomal fraction. The combined estrone-3-sulfatase activity in the other fractions (lysosomal, mitochondrial, and cytosolic fractions) is negligible and only accounts for < 5% of the total nuclear or microsomal sulfatase activity. Nuclear and microsomal estrone-3-sulfatases have different pH optima (pH 8.0 and 7.2, respectively). The apparent Km values for the nuclear and microsomal estrone-3-sulfatases are 2.5 and 10.1 microM, respectively, suggesting that the nuclear sulfatase has a considerably higher affinity for estrone-3-sulfate than the microsomal sulfatase. Moreover, the nuclear estrone-3-sulfatase is more sensitive to inhibition by several steroids than the microsomal sulfatase. The results suggest that estrone-3-sulfatase in the nuclear fraction is a different isozyme than that in the microsomal fraction.
Studies by several investigators have shown that 12-0-tetradecanoylphorbol-13-acetate (TPA) is an extraordinarily potent stimulator of differentiation of cultured human promyelocytic leukemia cells in vitro. In the present study, TPA was administered to humans by i.v. infusion without irreversible toxicity, and it was shown to have pharmacological activity for the treatment of myelocytic leukemia in patients refractory to cytosine arabinoside (Ara C), retinoic acid, and other antileukemic drugs. Marked decreases in bone marrow myeloblasts as well as temporary remission of disease symptoms were observed when TPA was administered alone or in combination with vitamin D3 and Ara C. Additional studies with TPA after the determination of optimum dosing regimens are needed to determine whether long-lasting or permanent remissions of myelocytic leukemia can be achieved. Transient and reversible side effects were observed after a 1-mg i.v. dose of TPA, but these adverse effects became less intense or disappeared when a lower dose of TPA was used. The results of this study indicate a therapeutic effect of TPA in patients with myelocytic leukemia.
Fifty-two patients with solid tumors had depressed white blood cell and neutrophil counts because of prior treatment with cytotoxic cancer chemotherapeutic drugs. These patients were given one or more i.v. infusions of 0.125-0.25 mg of 12-O-tetradecanoylphorbol-13-acetate (TPA), and this treatment increased the low white blood cell and neutrophil counts toward the normal range. The average white blood cell and neutrophil counts were 2.55 x 10(9)/liter and 1.76 x 10(9)/liter, respectively, before treatment with TPA. After one or more i.v. infusions of TPA, the white blood cell and neutrophil counts increased to peak values of 5. 92 x 10(9)/liter and 4.76 x 10(9)/liter, respectively, within a few days. Most patients had increased levels of white blood cells and neutrophils by 24 hr after a single i.v. infusion of 0.25 mg TPA. Elevated levels were observed for at least 3 days. This study demonstrates that treatment with parenteral TPA is feasible with useful biological activity. Only mild and reversible side effects were observed.
Administration of 0.5 or 1% lyophilized green tea (5 or 10 mg tea solids per ml, respectively) as the sole source of drinking fluid to female Long-Evans rats for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 30-37%, 15-27% and 26-60%, respectively. Oral administration of 0.5% lyophilized green tea to female CD-1 mice for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 33-37%, 12-22% and 172-191%, respectively. The in vitro addition of a green tea polyphenol mixture, a black tea polyphenol mixture or (-)-epigallocatechin gallate inhibited rat liver microsomal glucuronidation of estrone and estradiol in a concentration-dependent manner and their IC50 values for inhibition of estrogen metabolism were approximately 12.5, 50 and 10 microg/ml, respectively. Enzyme kinetic analysis indicates that the inhibition of estrone glucuronidation by 10 microM (-)-epigallocatechin gallate was competitive while inhibition by 50 microM (-)-epigallocatechin gallate was noncompetitive. Similarly, several flavonoids (naringenin, hesperetin, kaempferol, quercetin, rutin, flavone, alpha-naphthoflavone and beta-naphthoflavone) also inhibited rat liver microsomal glucuronidation of estrone and estradiol to varying degrees. Naringenin and hesperetin displayed the strongest inhibitory effects (IC50 value of approximately 25 microM). These two hydroxylated flavonoids had a competitive mechanism of enzyme inhibition for estrone glucuronidation at a 10 microM inhibitor concentration and a predominantly noncompetitive mechanism of inhibition at a 50 microM inhibitor concentration.
Cytochrome P450 enzymes that metabolize estrogens are expressed in the mammary gland, uterus, brain and other target tissues for estrogen action, and this results in the formation of hydroxylated estrogens in these tissues. Estradiol metabolites formed in target tissues at or near estrogen receptors may either be inactive or have important biological effects, and changes in the activities of estrogen-metabolizing enzymes in target tissues may profoundly influence estrogen action. Although some active estrogen metabolites exert hormonal effects in target tissues by interaction with the classical estrogen receptor, other metabolites appear to elicit unique biological responses that are not associated with activation of this receptor. Therefore, some of the many actions of estradiol may not be caused by estradiol per se, but may result from the formation of active estrogen metabolite(s) which function as local mediators or may activate their own unique receptors or effectors. This is an important area in need of more research. The present paper represents a review of the literature and perspectives by the authors on the functional role of estrogen metabolism in target tissues.
We evaluated the effects of a methanol extract from the leaves of the plant Rosmarinus officinalis L. (rosemary) on the metabolism and action of estradiol and estrone. Treatment of female CD-1 mice with 2% rosemary in AIN-76A diet for 3 weeks increased the liver microsomal 2-hydroxylation of estradiol and estrone by approximately 150%, increased their 6-hydroxylation by approximately 30% and inhibited the 16alpha-hydroxylation of estradiol by approximately 50%. Treatment of female CD-1 mice with 2% rosemary diet for 3 weeks also stimulated the liver microsomal glucuronidation of estradiol and estrone by 54-67% and 37-56%, respectively. In additional studies, feeding 2% rosemary diet to ovariectomized CD-1 mice for 3 weeks inhibited the uterotropic action of estradiol and estrone by 35-50% compared with animals fed a control diet. The results of this study showed that feeding female mice a 2% rosemary diet increased the liver microsomal oxidation and glucuronidation of estradiol and estrone and inhibited their uterotropic action.
In the present study, administration of green tea to SKH-1 mice, via the drinking fluid, was found to significantly reduce the incidence and volume of ultraviolet B (UVB) radiation-induced skin tumors. Thirty-six skin tumors induced by UVB and 32 skin tumors induced by UVB, in mice treated with green tea in their drinking water, were collected and examined for the presence of mutations in the p53 gene. Polymerase chain reaction products from p53 exons 5-8 were screened by single-strand conformation polymorphism and direct sequence analyses. Eight of 36 UVB-induced tumors contained nine p53 mutations, with four in exon 5 and five in exon 8. In contrast, nine of 32 UVB-induced tumors in mice treated with green tea contained 11 p53 mutations, with two in exon 5, five in exon 6 and four in exon 8. All of the p53 mutations occurred at dipyrimidine sequences. These results were further corroborated by p53 immunohistochemistry. The most frequent mutations were C-->T or T-->C transitions, which are consistent with the genetic alterations caused by UVB exposure. Interestingly, mutations found in exon 6 of the p53 gene occurred only in tumors from the UVB/green tea group. Thus, the tumors observed in UVB/green-tea-treated mice have a different exon distribution of p53 mutations than tumors obtained from mice treated with UVB alone.
Oral administration of green or black tea inhibited UVB light-induced complete carcinogenesis in the skin of SKH-1 mice. Green tea was a more effective inhibitor than black tea. Oral administration of decaffeinated green or black tea resulted in substantially less inhibitory activity than did administration of the regular teas, and in one experiment, administration of a high-dose level of the decaffeinated teas enhanced the tumorigenic effect of UVB. Oral administration of caffeine alone had a substantial inhibitory effect on UVB-induced carcinogenesis, and adding caffeine to the decaffeinated teas restored the inhibitory effects of these teas on UVB-induced carcinogenesis. In additional studies, topical application of a green tea polyphenol fraction after each UVB application inhibited UVB-induced tumorigenesis. The results indicate that caffeine contributes in an important way to the inhibitory effects of green and black tea on UVB-induced complete carcinogenesis.
Administration of 0.75% 2(3)-tert-butyl-4-hydroxyanisole (BHA) in AIN-76A diet to female CD-1 mice for 3 weeks increased liver microsomal glucuronidation of estradiol, estrone, 4-aminophenol, and 4-nitrophenol by 103, 187, 162, and 92%, respectively (at pH 7.4). The overall rate of NADPH-dependent metabolism of estradiol and estrone by liver microsomes of BHA-treated animals as determined by substrate disappearance was increased by 20-40% over that by liver microsomes from control animals. The rate of 2-hydroxylation of estradiol and estrone (the major metabolic pathway) was increased by 24-38%, the rate of formation of 6alpha-hydroxyestradiol plus 6beta-hydroxyestradiol was increased by 90-115%, and the rate of 6beta-hydroxyestrone formation (a minor metabolite formed in liver microsomes from control mice) was increased by approximately 370% over controls. In contrast, BHA administration had little or no effect on the liver microsomal formation of 4- and 16alpha-hydroxylated estradiol and estrone metabolites. Measurable levels of estradiol and estrone were observed in the serum and uterus of ovariectomized CD-1 mice at 30 min after a single i.p. injection of 100 or 300 ng of estradiol or estrone, and these levels were decreased by 30-60% in animals fed a 0.75% BHA diet for 18 days prior to the injection of estrogen. Feeding a 0.75% BHA-supplemented diet to ovariectomized CD-1 mice for 18 days inhibited the uterotropic effect of estradiol or estrone (45 or 75 ng/mouse, i.p. once daily for 3 days) as compared to the response of animals fed the control diet. BHA administration also inhibited estradiol- or estrone-stimulated [3H]thymidine incorporation into uterine DNA. In conclusion, feeding a 0.75% BHA-supplemented diet to female CD-1 mice for 2-3 weeks increased the activities of liver microsomal enzymes that catalyze uridine 5'-diphosphoglucuronic acid-dependent glucuronidation and NADPH-dependent oxidation of estradiol and estrone, enhanced the in vivo metabolism of these estrogens, and inhibited their uterotropic action.
The effects of topical applications of very low doses of curcumin (the major yellow pigment in turmeric and the Indian food curry) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidation of DNA bases in the epidermis and on tumor promotion in mouse skin were investigated. CD-1 mice were treated topically with 200 nmol of 7,12-dimethylbenz[a]anthracene followed one week later by 5 nmol of TPA alone or together with 1, 10, 100 or 3000 nmol of curcumin twice a week for 20 weeks. Curcumin-mediated effects on TPA-induced formation of the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HMdU) and tumor formation were determined. All dose levels of curcumin inhibited the mean values of TPA-induced HMdU formation in epidermal DNA (62-77% inhibition), but only the two highest doses of curcumin strongly inhibited TPA-induced tumor promotion (62-79% inhibition of tumors per mouse and tumor volume per mouse). In a second experiment, topical application of 20 or 100 nmol (but not 10 nmol) of curcumin together with 5 nmol TPA twice a week for 18 weeks markedly inhibited TPA-induced tumor promotion. Curcumin had a strong inhibitory effect on DNA and RNA synthesis (IC50 = 0.5-1 microM) in cultured HeLa cells, but there was little or no effect on protein synthesis.
Female CD-1 mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate. Mice with established papillomas were then treated with black tea or decaffeinated black tea (approximately 4 mg tea solids/ml) as the sole source of drinking fluid for 11-15 weeks. In four separate experiments, oral administration of black tea inhibited the growth of papillomas (increase in tumor volume/mouse) by an average of 35%, 37%, 41% and 48%, respectively. Studies with decaffeinated black tea gave inconsistent results. In one experiment, administration of decaffeinated black tea inhibited papilloma growth (increase in tumor volume/mouse) by 27%, but in two additional experiments papilloma growth was stimulated by 14% and 193%, respectively. In a separate experiment, skin tumors were generated by treating SKH-1 female mice with ultraviolet B light (UVB; 30 mJ/cm2) twice weekly for 22 weeks, after which UVB administration was stopped. Tumors were allowed to develop during the following 13 weeks, and tumor-bearing mice were then treated with black tea (6 mg/ml tea solids) as the drinking fluid for 11 weeks. In this experiment, tumor growth (increase in tumor volume/mouse) was inhibited by 70%. Histological examination revealed that tea-treated mice had a 58% decrease in the number of nonmalignant tumors (primarily keratoacanthomas)/mouse and a 54% decrease in the number of squamous cell carcinomas/mouse. In addition, administration of black tea decreased the volume per tumor by 60% for nonmalignant tumors and by 84% for carcinomas. Mechanistic studies with tumors from these mice revealed that administration of black tea decreased the bromodeoxyuridine labeling index in squamous cell papillomas, keratoacanthomas and squamous cell carcinomas by 56%, 45% and 35%, respectively, and the apoptosis index was increased by 44%, 100% and 95%, respectively. Administration of black tea decreased the mitotic index in keratoacanthomas and squamous cell carcinomas by 42% and 16%, respectively. The results indicate that oral administration of black tea to tumor-bearing mice inhibited proliferation and enhanced apoptosis in nonmalignant and malignant skin tumors.
Topical application of curcumin inhibits chemically induced carcinogenesis on mouse skin, and oral administration of curcumin inhibits chemically induced oral, forestomach, duodenal, and colon carcinogenesis. Curcumin and other inhibitors of cyclooxygenase and lipoxygenase are thought to inhibit carcinogenesis by preventing the formation of arachidonic acid metabolites. In contrast to our expectation of a tumorigenic effect of arachidonic acid, we found that treatment of 7,12-dimethylbenz[a]anthracene-initiated mouse skin with very high doses of arachidonic acid twice daily, 5 days a week for 26 weeks, failed to result in tumors. We considered the possibility that some of the cancer chemopreventive effects of curcumin may be related to an effect of this compound on cellular differentiation, and we investigated the effect of curcumin on differentiation in the human promyelocytic HL-60 leukemia cell model system. Although curcumin alone had little or no effect on cellular differentiation, when it was combined with all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 a synergistic effect was observed. It is possible that many dietary chemicals in fruits, vegetables, and other edible plants can prevent cancer by synergizing with endogenously produced stimulators of differentiation such as all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and butyrate. More research is needed to test this hypothesis. Administration of green or black tea inhibits carcinogenesis in several animal models, and tumor growth is also inhibited. Several examples were presented of chemopreventive agents that inhibit carcinogenesis in one animal model but enhance carcinogenesis in a different animal model. Greater efforts should be made to understand mechanisms of cancer chemoprevention and to determine whether a potential chemopreventive agent is useful in many experimental settings or whether it is useful in only a limited number of experimental settings.
Treatment of human promyelocytic leukemia HL-60 cells with 10 muM curcumin for 48 h inhibited cellular proliferation and induced small increases in differentiation (100-200%) as measured by the proportion of cells that reduced nitroblue tetrazolium (NBT) and expressed Mac-1. Synergistic induction of differentiation as measured by the above markers was observed when 1-10 muM curcumin was combined with 10-100 nM all-trans retinoic acid (RA) or with 100 nM 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3). Cell morphology and flow cytometric studies (with the monocytic surface antigen CD14) indicated that combinations of RA and curcumin stimulated differentiation predominantly to granulocytes whereas combinations of vitamin D3 and curcumin stimulated differentiation predominantly to monocytes. Studies on cell cycle kinetics indicated that treatment of HL-60 cells with a combination of RA and curcumin for 48 or 96 h reduced the proportion of cells in the S phase of the cell cycle and increased the proportion of cells in the G0/G1 phase of the cell cycle to a greater extent than occurred for cells treated with either compound alone. Combinations of vitamin D3 and curcumin did not alter cell cycle kinetics to a greater extent than was observed for either compound alone. Combinations of RA and curcumin or vitamin D3 and curcumin inhibited the proliferation of HL-60 cells to a greater extent than was observed for either compound alone. The results indicate that curcumin is a weak stimulator of differentiation in HL-60 cells and that is has synergistic effects when combined with RA or vitamin D3. Combinations of curcumin and RA have a particularly potent inhibitory effect on the proliferation of HL-60 cells.
Chinese hamster V-79 cells were treated with high cytotoxic or low noncytotoxic concentrations of the highly carcinogenic and mutagenic (-)-(1R,2S,3S,4R)-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [(-)-B[c]PhDE; fjord-region diol epoxide] or its biologically less active (+)-(1S,2R,3R,4S) enantiomer [(+)-B[c]PhDE]. The benzylic 4-hydroxyl group and the epoxide oxygen are trans in both enantiomers. Independent 8-azaguanine-resistant clones were isolated. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene was amplified by reverse transcription-PCR and sequenced. For (-)-B[c]PhDE, mutation frequencies were 10- or 356-fold above background for the low (0.01-0.1 microM; 97% cell survival) or high (1.0-1.25 microM; 26% cell survival) doses, respectively. For the high dose group, 20 of 64 base substitutions occurred at GC base pairs (31%) and 44 at AT base pairs (69%). For the low-dose group, 6 of 55 base substitutions were at GC base pairs (11%), and 49 were at AT base pairs (89%). For the less active (+)-B[c]PhDE, mutation frequencies were 17- or 372-fold above background for the low (0.12-0.5 microM; 95% cell survival) or high (2.0-3.0 microM; 31% cell survival) doses, respectively. In contrast to the results with the (-)-B[c]PhDE, both the high- and the low-dose groups for (+)-B[c]PhDE gave a 50:50 distribution of base substitution at GC versus AT base pairs. Our data indicate that: (a) transversions were the predominant base substitutions observed for both the (+)- and (-)-enantiomers of B[c]PhDE; (b) (-)-B[c]PhDE showed high selectivity for causing AT --> TA transversions, whereas considerably less selectivity was observed for (+)-B[c]PhDE; (c) (-)-B[c]PhDE had a different hot spot profile for base substitutions than did (+)-B[c]PhDE, but some common hot spots were observed for both compounds; and (d) decreasing the dose of (-)-B[c]PhDE increased the proportion of mutations at AT base pairs and decreased those at GC base pairs, but this was not observed for (+)-B[c]PhDE.
In this study, we evaluated the effects of pH, in vitro inhibitors, in vivo enzyme inducers, age, and sex on the glucuronidation of estradiol and estrone by rat liver microsomes. Although the pH dependence curves for the glucuronidation of estradiol and estrone were similar, the pH dependence curves for these estrogens by liver microsomes from adult male rats were very different from those by liver microsomes from adult female rats. These results suggest that liver microsomes from adult male and have different estrogen glucuronosyltransferases. Liver microsomes from immature or adult female rats catalyzed the glucuronidation of estrone and estradiol more rapidly than liver microsomes from age-matched male rats. Intraperitoneal injection of sodium phenobarbital (75 mg/kg/day) or dexamethasone (75 mg/kg/day) into immature or adult male or female rats for 3-4 days resulted in a 33-58% increase in liver microsomal glucuronosyltransferase activity for estradiol, but there was little or no stimulatory effect on glucuronosyltransferase activity for estrone. Treatment of immature or adult male or female rats with 3-methylcholanthrene (25 mg/kg/day) for 3-4 days did not stimulate liver microsomal glucuronosyltransferase activity for estradiol or estrone, but the glucuronidation of 4-nitrophenol was stimulated several-fold. The in vitro addition of testosterone had a strong inhibitory effect on the glucuronidation of estradiol and estrone by liver microsomes from both adult male and female rats, whereas the in vitro addition of 4-nitrophenol had a slightly greater inhibitory effect on the glucuronidation of estradiol and estrone by adult male liver microsomes than by adult female liver microsomes. In conclusion, our results suggest that male and female rat livers have different estrogen glucuronosyltransferases and that the glucuronidation of estradiol, estrone, and 4-nitrophenol is catalyzed by different glucuronosyltransferases that are under different regulatory control.