Eero Mäntylä

Simple methods of quantifying oxidation products and antioxidant potential of low density lipoproteins

OBJECTIVES The present study describes new methods for the measurement of oxidation products and antioxidant potential of low density lipoproteins (LDL). DESIGN AND METHODS LDL is isolated by precipitation with buffered heparin. The assay for LDL oxidation products (LDL-BDC) is based on determination of baseline levels of conjugated dienes (BDC) in lipids extracted from LDL. The assay for antioxidant potential of LDL (LDL-TRAP) is based on the ability of LDL to trap peroxyl radicals. RESULTS LDL-BDC was found to increase linearly over a range from 100 to 1750 microL, LDL-TRAP from 250 to 1750 microL of serum taken for precipitation. For LDL-BDC, the CV was 4.4% and 4.5% for within- and between-assay precision, respectively. For the LDL-TRAP, the CV was 8.1% and 8.7% for within- and between-assay precisions, respectively. Freezing of the serum (2 weeks at -70 degrees C) did not affect LDL-BDC or LDL-TRAP levels. A negative correlation was found to exist between the LDL-BDC and LDL-TRAP values. LDL-BDC and LDL-TRAP values were at the same level in both sexes. The LDL-BDC was found to increase with age. Short-term intervention with antioxidants increased LDL-TRAP substantially, but did not affect the LDL-BDC level. CONCLUSIONS These methods are fast and simple to perform, and can, therefore, be applied to clinical purposes.

Toxicity of Antiestrogens

The object of this article is to review briefly the preclinical and clinical safety of some antiestrogens. Tamoxifen, toremifene, droloxifene, and idoxifene are polyphenylethylene antiestrogens, whereas the pure antiestrogen, ICI 182,780 or faslodex, as well as raloxifene, is of a different structure. Tamoxifen has been shown to be genotoxic in several studies. It induces unscheduled DNA synthesis in rat hepatocytes and micronuclei in MCL‐5 a cells in vitro. Tamoxifen also induces aneuploidy in rat liver in vivo and chromosome aberrations and micronuclei in mouse bone marrow. Toremifene has also shown to be genotoxic, but to a far lower extent, by inducing micronuclei in MCL‐5 a cells in vitro and by inducing aneuploidy in rat liver in vivo. Tamoxifen has been shown to be hepatocarcinogenic in the rat in at least four independent long‐term studies. The initiation of tumors in the rat is the result of metabolic activation by cytochrome P450 isoenzymes to an electrophile(s) that binds irreversibly to DNA. The other antiestrogens have not been shown to be carcinogenic in rodents. In several independent clinical studies, the risk of endometrial cancer has increased among tamoxifen‐treated women. After reviewing the available data, the International Agency for Research on Cancer concluded that there was sufficient evidence to show that tamoxifen is a class I human carcinogen. The increased risk for endometrial cancer occurs predominantly among women who are 50 years old or older and who have been treated with tamoxifen. It is not yet clear whether the uterine tumor formation is a result of genetic mechanisms, analogous to those seen in the rat liver or due to the estrogen agonist action of tamoxifen. However, the other antiestrogens with a more or less similar intrinsic estrogenic potential have not been shown to be carcinogenic in humans.

A TWO-YEAR DIETARY CARCINOGENICITY STUDY OF THE ANTIESTROGEN TOREMIFENE IN SPRAGUE-DAWLEY RATS

The carcinogenic potential of the nonsteroidal triphenylethylene antiestrogen toremifene (Fareston) was evaluated in a standard 104-week rat dietary carcinogenicity study. The doses were 0, 0.12, 1.2, 5.0 and 12 mg/kg/day and the number of animals 50/sex/dose group. The body weight gain and food consumption were monitored once weekly (study weeks 1-16) or once every four weeks thereafter (study weeks 17-104). Blood samples were taken at weeks 34, 52 and 104 and the plasma concentrations of toremifene, as well as the two main metabolites (deaminohydroxy)toremifene and N-demethyltoremifene, were measured. All doses of toremifene reduced food intake and body weight gain. Toremifene caused a significant reduction in mortality, which was mainly due to reduced incidences of pituitary tumors. This was evident in all dose groups. Drug-related decrease of mammary tumors in females (at all doses) and testicular tumors in male rats (doses > or = 1.2 mg/kg/day) were also evident. The incidence of the preneoplastic foci of basophilic hepatocytes were significantly decreased in treated female groups. Toremifene induced no preneoplastic or neoplastic lesions. Based on histopathology, no obvious toxicity could be observed. Drug-related changes were observed in the genital organs, thyroid, spleen, mammary gland, adrenal, kidney, stomach and lung. These changes were due to hormonal disturbances or as a result of reduced food consumption or reduced incidences of pituitary, mammary or testicular tumors. This study indicates that toremifene is an efficient antiestrogen in long-term treatment, is well tolerated and has no tumorigenic potential in rats.

ANTIOXIDANT PROPERTIES OF THE TRIPHENYLETHYLENE ANTIESTROGEN DRUG TOREMIFENE

Abstract The aim of the present study was to investigate antioxidativity of the triphenylethylene antiestrogen toremifene. Toremifene and its structural analogues were studied for their ability to inhibit chain reactions of lipid peroxidation and to act as scavengers of free radicals in vitro, and the effects of toremifene were compared to those of the estrogens, tamoxifen and known antioxidants. Moreover, the in vivo antioxidativity of toremifene was tested in a long-term experiment with rats. The ability of toremifene to prevent lipid peroxidation was assayed in two different test systems. In the first assay (initiated with ascorbate/ADP-FeCl3, detection by the formation of TBA-reactive material) toremifene was found to act as an efficient membrane antioxidant with an IC50-value (18 μM) comparable to that of tamoxifen (26 μM) and α-tocopherol (43 μM). Toremifene derivatives 4-hydroxytoremifene (IC50 = 8 μM) and Fc 1159 (IC50 = 31 μM), as well as diethylstilbestrol (IC50 = 17 μM) were also active while estradiol showed only weak antioxidativity (IC50 = 300 μM) in this test system. In the other assay (peroxidation initiated with t-butylhydroperoxide, detection by luminol-enhanced chemiluminescence) toremifene prevented lipid peroxidation only at high concentrations (IC50 = 450 μM) but the metabolite 4-hydroxytoremifene (IC50 = 0.18 μM), estradiol (IC50 = 4.6 μM) and diethylstilbestrol (IC50 = 1.7 μM) showed potent antioxidant activity. The potency of 4-hydroxytoremifene even exeeded that of α-tocopherol (IC50 = 2.0 μM) and butylated hydroxyanisole (IC50 = 1.1 μM). Toremifene was found to have some superoxide anion but no peroxyl radical scavenging activity. Interestingly, diethylstilbestrol turned out to be a potent scavenger of peroxyl radicals. Treatment of female Sprague-Dawley rats with toremifene (12 or 48 mg/kg) was found to decrease serum levels of lipid peroxides. This was seen at various time points (2 days, 5 weeks, 6 and 12 months) during long-term administration of toremifene to rats, and results obtained with two different methods (diene conjugation, TBA-reactive material) gave similar results. The present study thus showed that (i) like steroidal estrogens and tamoxifen toremifene is a potent membrane antioxidant in vitro, (ii) the antioxidant action of toremifene is not due to scavenging of free radicals and, importantly, (iii) toremifene acts antioxidatively also in living organisms in vivo.

DNA ADDUCT FORMATION BY TAMOXIFEN AND STRUCTURALLY-RELATED COMPOUNDS IN RAT LIVER

Abstract Binding of diethylstilbestrol and four different triphenylethylene derivatives: tamoxifen, toremifene, clomiphene and triparanol to DNA in rat liver, was studied using the 32P-postlabelling method with HPLC-radioactivity detection. Three different modifications of the 32P-postlabelling technique (a) a bisphosphate method with adduct enrichment by nuclease P1 (NP1)-treatment or (b) by butanol extraction and (c) a monophosphate method, were applied in order to provide an unbiased analysis of adduct formation. When tamoxifen was administered by daily gavage for 4 weeks (80 μmol/kg for 2 weeks and 40 μmol/kg for a further 2 weeks) two major adducts and about six minor adducts were produced in the liver of female Sprague-Dawley rats. Equimolar doses of toremifene produced one apparent adduct. The adduct levels in the tamoxifen and toremifene treated rats were 600 and 2/108 nucleotides, respectively. Under conditions used, clomiphene, triparanol and diethylstilbestrol did not produce DNA adducts. The present and previous data suggest that modification (a) is the 32P-postlabelling method of choice for risk assessment in human subjects. Modification (c) with butanol extraction after labelling has the advantage of low background radioactivity and may be preferable if large amounts of DNA are available. The main tamoxifen adducts were suggested to be α-(N2-deoxyguanosinyl)tamoxifen and α-(N2-deoxyguanosinyl)-N-desmethyltamoxifen.

DNA binding of tamoxifen and its analogues: Identification of the tamoxifen-DNA adducts in rat liver

DNA binding of tamoxifen and some structurally-related drugs (toremifene, clomiphene, triparanol and raloxifene) in rat liver was studied using the 32P-postlabelling method. As only tamoxifen was shown to form high levels of DNA adducts, the identity of these adducts was studied. Recently, we have identified by mass spectroscopy the two main tamoxifen adducts in rat liver DNA as the N-desmethyltamoxifen and tamoxifen adducts of N2-deoxyguanosine in which the linkage is through alpha-carbon in the tamoxifen structure. Minor adducts were identical to different diastereomers of alpha-(N2-deoxyguanosinyl)tamoxifen and of alpha-(N6-deoxyadenosinyl)tamoxifen. Altogether these adducts accounted for at least 95% of adducts formed in vivo, implicating that the alpha-hydroxylation of the ethyl group is the major activation pathway for DNA adducts.

Induction of Endometrial Cancer by Tamoxifen in the Rat

Induction of preneoplastic and neoplastic changes by two antiestrogenic drugs was studied in female rats. Groups of female rats were treated with tamoxifen citrate (TAM) (11.3 or 45 mg/kg) or toremifene citrate (TOR) (3, 12, or 48 mg/kg) by daily oral gavage to rats. Also, a group of animals was treated for 20–52 weeks followed by a 12–13 recovery period. The animals were killed 13–52 weeks after treatment, including control rats. The uteri of all the rats were examined macroscopically and histopathologically. No pathological uterine changes were detected in the control group, the three TOR groups, or the low-dose TAM group. However, in the high-dose TAM group, 10 cases (10/104; 9.6%) of squamous cell metaplasia were detected. In three (3/104; 2.9%) of the metaplasias, a focal dysplastic change was observed. In two (2/104; 1.9%) of these, focal invasive squamous cell carcinomas were verified. The present data on endometrial cancer induced by TAM provides a link between human and animal data and suggests that a similar mechanism of cancer induction may be at work in both species.

61 Endometrial cancer induction by tamoxifen in the rat

Tamoxifen (TAM) is an antiestrogenic breast cancer drug. Medication with TAM increases the risk of secondary cancers in the endometrium. In the rat TAM is a strong hepatocarcinogen. Toremifene (TOR), a new antiestrogen clinically as effective as TAM, has not shown any hepatic or endometrial adverse effects. We studied the effects of TAM and TOR on rat endometrium. Three separate studies were put together. Female SDrats were daily treated with vehiche or with the drugs at equimolar doses (20 or 80 J1.mol/kg) p.o. for 13-52 weeks. Both drugs produced comparable uterine atrophy and weight reduction at both dose levels indicating an equal hormonal effect. No preneoplastic or neoplastic changes were observed in contral or TOR groups or in the low-dose TAM group. The incidence of squamous cell metaplasia with prominent keratinization was 10% in the high-dose TAM group. In 3 of the metaplasias there was a focal dysplastic change and two of these animals beared also a focal invasive squamous cell carcinoma. The carcinomas were found after 20 or 26 weeks of dosing, in both cases after a recovery period. The histopathological outlook of these lesions argued against a mere hormonal etiology. TOR produced no lesions although the estrogen antagonist/agonist activities and the endometrial proliferative potentials of these two drugs at the dose levels used are closely comparable. In conclusion, a nonhormonal (possibly genotoxic) mechanism in the TAM-induced endometrial carcinogenesis is probable. TOR might be more safe clinically, especially when healthy women are treated with antiestrogens in breast cancer preventive indication.

60 Tamoxifen and Toremifene: The beneficial hypolipemic effect in women is mediated by cholesterol biosynthesis inhibition

Tamoxifen (TAM) is an effective antiestrogenic anticancer drug. In longterm therapy it decreases serum cholesterol and LDL-cholesterollevels and decreases the risk of coronary artery disease. The mechanism of the hypolipemic effect has not been fully elucidated Toremifene (TOR) is a new antiestrogenic anticancer drug that has clinical efficacy equal to that of TAM. The structure of TOR is closely related to that of TAM and animal experiments suggest that also TOR can have hypolipemic effects. The effect of TAM and TOR therapy on serum lipid levels were studied in postmenopausal advanced breast cancer patients. The effect on cholesterol biosynthesis was evaluated by measuring cholesterol precursor levels by gas-liquid chromatography at pre-dose and after 2, 6 or 12 months therapy. Both drugs decreased cholesterol and LDL-cholesterol levels almost equally. This suggests that also TOR can be expected to have TAM-like beneficial antiatherogenic effect during long-term therapy. Of the cholesterol precursors Δ 8 -cholestenollevel was increased up to about 50-fold with both drugs; in other precursor levels only minor changes were seen. This indicates that these antiestrogens inhibit Δ 8 -cholestenol conversion to lathosterol and as a result the cholesterol biosynthesis is downregulated. This inhibition is suggested to be the main method for the hypolipemic effect of these drugs.