Protective roles of quinone reductase and tamoxifen against estrogen-induced mammary tumorigenesis.

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Sulfatase activity in the oyster Crassostrea virginica: its potential interference with sulfotransferase determination.

Two sulfatase isoforms, a soluble one with an optimum pH of 5.0, and a microsomal one with an optimum pH of 7.6, were observed in digestive gland, gonads, mantle and gills of the oyster C. virginica. The highest sulfatase activity was recorded in the digestive gland cytosol and is likely to interfere with the in vitro determination of sulfotransferase activity. Indeed, the sulfatase inhibitor Na(2)SO(3) led to an increase of measured sulfotransferase activity (31+/-9%), suggesting that those sulfatases might be partially responsible for the low sulfotransferase activities found in C. virginica.

The effects of steroidal estrogens in ACI rat mammary carcinogenesis: 17beta-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol, 16alpha-hydroxyestradiol, and 4-hydroxyestrone.

Several investigators have suggested that certain hydroxylated metabolites of 17beta-estradiol (E2) are the proximate carcinogens that induce mammary carcinomas in estrogen-sensitive rodent models. The studies reported here were designed to examine the carcinogenic potential of different levels of E2 and the effects of genotoxic metabolites of E2 in an in vivo model sensitive to E2-induced mammary cancer. The potential induction of mammary tumors was determined in female ACI rats subcutaneously implanted with cholesterol pellets containing E2 (1, 2, or 3 mg), or 2-hydroxyestradiol (2-OH E2), 4-hydroxyestradiol (4-OH E2), 16alpha-hydroxyestradiol (16alpha-OH E2), or 4-hydoxyestrone (4-OH E1) (equimolar to 2 mg E2). Treatment with 1, 2, or 3 mg E2 resulted in the first appearance of a mammary tumor between 12 and 17 weeks, and a 50% incidence of mammary tumors was observed at 36, 19, and 18 weeks respectively. The final cumulative mammary tumor incidence in rats treated with 1, 2, or 3 mg E2 for 36 weeks was 50%, 73%, and 100% respectively. Treatment of rats with pellets containing 2-OH E2, 4-OH E2, 16alpha-OH E2, or 4-OH E1 did not induce any detectable mammary tumors. The serum levels of E2 in rats treated with a 1 or 3 mg E2 pellet for 12 weeks was increased 2- to 6-fold above control values (approximately 30 pg/ml). Treatment of rats with E2 enhanced the hepatic microsomal metabolism of E2 to E1, but did not influence the 2- or 4-hydroxylation of E2). In summary, we observed a dose-dependent induction of mammary tumors in female ACI rats treated continuously with E2; however, under these conditions 2-OH E2, 4-OH E2, 16alpha-OH E2, and 4-OH E1 were inactive in inducing mammary tumors.

Pharmacological study of Stachytarpheta cayennensis Vahl in rodents.

Freeze-dried aqueous extracts (AEs, 0.1-1g/kg body wt., p.o.) obtained from entire or selected parts of Stachytarpheta cayennensis were tested for their effects on gastric secretion, gastric motility, inflammation and pain in rodents, with the purpose of validating the plant's ethnomedical uses. The AE-Total, AE-Flowers and AE-Leaves but not AE-Stems inhibited the gastric acid secretion in pylorus-ligated rats with varying potency. Purification of AEs yielded the semipurifed fractions EtFs rich in iridoids. All the EtFs with exception of EtF-Stems inhibited gastric acid secretion of pylorus ligated mice. While AE-Total stimulated the intestinal transit of mice by 43%, AE-Leaves delayed it by 38%. These effects on intestinal transit were not observed when the EtFs were tested. Only AE-Leaves and AE-Flowers altered the gastric emptying of semisolids, increasing it by 45% and 69%, respectively. These results indicate that the compounds related to inhibition of gastric acid secretion and gastrointestinal motility are different. The AE-Total reduced abdominal writhing induced by acetic acid potently (ED50 value = 700 mg/kg, p. o.) without altering the writhes induced by acetylcholine. Attempts to identify the mechanism of analgesia were unsuccessful since the AE-Total did not show analgesic effects when tested in different models of pain such as formalin and capsaicin or the tail-flick test. Pretreatment of animals with AE-Total did not show antiinflammatory activity in any of the acute (paw edema induced by carrageenin, dextran or histamine, pleurisy induced by carrageenin and capsaicin-induced mouse ear edema) or chronic (air pouch) models used. No toxic signs were observed after administration of the different extracts up to 2 g/kg body wt., p.o. Collectively, the results confirmed folk information indicating presence of analgesic, mild laxative and potent inhibition of gastric secretion activities in the aqueous extracts of S. cayennensis. The results do not, however confirm the folk use of the plant as an antiinflammatory medicine.

Inhibition of rat liver sulfotransferases SULT1A1 and SULT2A1 and glucuronosyltransferase by dietary flavonoids.

1. Dietary flavonoids including kaempferol, quercetin, genistein and daidzein were tested for their ability to alter the conjugation of oestradiol (E(2)) via rat liver sulfotransferases and glucuronosyltransferase. 2. All four flavonoids inhibited the sulfonation of E(2) via phenol sulfotransferase, SULT1A1 with IC(50)s ranging from 0.29 to 4.61 micro M. Sulfonation of dehydroisoandrosterone (DHEA) via hydroxysteroid sulfotransferase, SULT2A1, was inhibited by higher amounts of the flavonoids (IC(50)s ranging from 34 to 116 micro M). 3. All flavonoids inhibited the formation of E(2)-beta-glucuronides (at carbon atoms 3 and 17) with IC(50)s ranging from 43 to 260 micro M. Glucuronidation of 4-methylumbelliferone (4-MU) was inhibited by high amounts of the flavonoids (IC(50)s ranging from 860 to 1550 micro M). 4. Hydrolysis of sulfonated oestrogens via arylsulfatase-c (ARSC) or 4-methylumbelliferone beta-glucuronidate (MUG) were not inhibited by the flavonoids. 5. It is concluded that SULT1A1 but not SULT2A1 or glucuronosyltransferase is highly sensitive to inhibition by dietary flavonoids. The potency of the inhibition for SULT1A1 (quercetin > kaempferol > genistein > daidzein) suggests a dependency on the number and position of hydroxyl radicals in the flavonoid molecule.

Solanum paniculatum L. (Jurubeba): potent inhibitor of gastric acid secretion in mice.

Solanum paniculatum L. is used commonly in Brazilian folk medicine for the treatment of liver and gastrointestinal disorders. The freeze-dried aqueous extracts (WEs) obtained from distinct parts of the plant (flowers, fruits, leaves, stems and roots) were tested to determine their antiulcer and antisecretory gastric acid activities using mice. The aqueous extracts of roots, stems and flowers inhibited gastric acid secretion in pylorus-ligated mice with ED50 values of 418, 777 and 820 mg/kg body wt. (i.d.), respectively. Extracts of leaves (0.5-2 g/kg body wt., i.d.) did not affect gastric secretion, whereas fruit extracts (0.5-2 g/kg body wt., i.d.) stimulated gastric acid secretion. The stimulatory effect of the fruit extract was inhibited by pretreatment with atropine (5 mg/kg body wt., i.m.) but not with ranitidine (80 mg/kg body wt., i.p.) suggesting that the fruit extract activates the muscarinic pathway of gastric acid secretion. In contrast, administration of the root extract into the duodenal lumen inhibited histamine- and bethanechol-induced gastric secretion in pylorus-ligated mice. In addition, the aqueous extract of roots (ED50 value, 1.2 g/kg body wt., p.o.) protected the animals against production of gastric lesions subsequent to the hypersecretion induced in mice by stress following cold restraint. This effect was not reproduced when the lesions were induced by blockade of prostaglandins synthesis via subcutaneous injection of indomethacin. Thus, antiulcer activity of the plant extracts appears to be related directly to a potent anti-secretory activity. No toxic signs were observed following administration of different extracts up to 2 g/kg body wt., p.o. Collectively, the results validate folk use of Solanum paniculatum L. plant to treat gastric disorders.

Natural products isolated from Mexican medicinal plants: novel inhibitors of sulfotransferases, SULT1A1 and SULT2A1.

Calophyllum brasiliense, Lonchocarpus oaxacensis, and Lonchocarpus guatemalensis are used in Latin American folk medicine. Four natural xanthones, an acetylated derivative, and two coumarins were obtained from C. brasiliense. Two flavanones were extracted from L. oaxacensis and one chalcone from L guatemalensis. These compounds were tested as substrates and inhibitors for two recombinant sulfotransferases (SULTs) involved in the metabolism of many endogenous compounds and foreign chemicals. Assays were performed using recombinant phenolsulfotransferase (SULT1A1) and hydroxysteroidsulfotransferase (SULT2A1). Three of the five xanthones, one of the flavonoids and the coumarins tested were substrates for SULT1A1. None of the xanthones or the flavonoids were sulfonated by SULT2A1, whereas the coumarin mammea A/BA was a substrate for this enzyme. The natural xanthones reversibly inhibited SULT1A1 with IC50 values ranging from 1.6 to 7 microM whereas much higher amounts of these compounds were required to inhibit SULT2A1 (IC50 values of 26-204 microM). The flavonoids inhibited SULT1A1 with IC50 values ranging from 9.5 to 101 microM, which compared with amounts needed to inhibit SULT2A1 (IC50 values of 11 to 101 microM). Both coumarins inhibited SULT1A1 with IC50 values of 47 and 185 pM, and SULT2A1 with IC50 values of 16 and 31 microM. The acetylated xanthone did not inhibit either SULT1AI or SULT2A1 activity. Rotenone from a commercial source had potency comparable to that of the flavonoids isolated from Lonchocarpus for inhibiting both SULTs. The potency of this inhibition depends on the position and number of hydroxyls. The results indicate that SULT1A1, but not SULT2A1, is highly sensitive to inhibition by xanthones. Conversely, SULT2A1 is 3-6 times more sensitive to coumarins than SULT1A1. The flavonoids are non-specific inhibitors of the two SULTs. Collectively, the results suggest that these types of natural products have the potential for important pharmacological and toxicological interactions at the level of phase-II metabolism via sulfotransferases.

Challenges of cancer drug design: a drug metabolism perspective.

The time course and duration of action of drugs used in cancer chemotherapy are greatly influenced by the molecular and biochemical properties of enzymes associated with their metabolism. Variation in the response of individual patients to cancer chemotherapeutic agents is in large measure due to genetic and environmental factors that impinge on specific enzymes belonging to the two major classes of drug metabolizing enzymes. Current knowledge of the molecular biology and biochemistry of phase I drug metabolizing enzymes (cytochrome P450, flavin-containing and xanthine oxidases, NADPH quinone reductase, and aldehyde and dihydropyridine dehydrogenases), and phase II enzymes (glucuronosyl-, sulfo-, N-acetyl-, and glutathione transferases, and hydrolases) is reviewed briefly. Advances in understanding genetic and environmental factors that influence activities of phase I and phase II pathways of drug metabolism are discussed in the first sections of this review followed by a consideration of the influence of drug metabolism on the actions of agents currently used in the treatment of cancer. Emphasis is given to drugs that have recently been introduced into the armamentarium of cancer chemotherapy including: inhibitors of chromatin function, target-based inhibitors of signal transduction and cyclin-dependent kinases, and angiogenesis inhibitors acting on metalloproteinases, epithelial cell growth, angiogenesis stimulation, and endothelial-specific integrins.