C.K. Lim

Chemoprevention of breast cancer by tamoxifen : Risks and opportunities

ABSTRACT The antiestrogen tamoxifen is widely used in the adjuvant therapy of breast cancers in women and helps to prevent the occurrence of breast tumors in healthy women. However, epidemiological studies have shown tamoxifen treatment to be associated with a 2- to 5-fold increased risk of endometrial cancer. In rats but not in mice, long-term administration of tamoxifen results in an increase in hepatocellular carcinomas. Mechanistically, this occurs through metabolic activation of the drug, mainly by the CYP3A family, to an electrophilic species, that causes DNA damage in target tissues, and subsequently leads to gene mutations. It is controversial whether low levels of DNA damage occur in human uterine tissues, and there is no evidence that this can be causally related to the mechanisms of carcinogenesis. In healthy women, the risk:benefits for the use of tamoxifen is in part related to the risk of developing breast cancer. The results from the carcinogenicity studies in rats do not predict the likelihood that women will develop liver cancer or indeed cancers in other organs. The mechanism of endometrial cancer in women remains unresolved, but the experience with tamoxifen has highlighted the potential problems that need to be addressed in the assessment of future generations of selective estrogen receptor modulators.

Determination of quercetin in human plasma by HPLC with spectrophotometric or electrochemical detection

A reversed-phase high-performance liquid chromatographic method for the determination of quercetin in human plasma following intravenous infusion is described. Quercetin in plasma was extracted with methanol-dimethyl sulphoxide (4:1 v/v) and separated on a C18 Hypersil-BDS column with 44% (v/v) methanol in 0.1 M ammonium acetate (pH 5.15) containing 0.27 mM EDTA as the mobile phase. The drug was detected specifically and sensitively at its absorption maximum of 375 nm, or electrochemically, with a detection limit of 80 ng/mL and 2 ng/mL, respectively.

Identification of photochemical oxidation products of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin by on-line high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Abstract An on-line HPLC-electrospray ionization tandem mass spectrometry system has been developed for the separation and characterization of the photochemical oxidation products of 5,10,15,20-tetra( m -hydroxyphenyl)chlorin ( m -THPC), a new photodynamic therapeutic agent. m -THPC was photo-oxidized in methanol solution by exposure to white laboratory light for one week. The products were then separated by isocratic elution on an Apex C 18 column with 0.1% trifluoroacetic acid in acetonitrile (23:77, v/v) as mobile phase and characterized on-line by electrospray tandem mass spectrometry. The results showed the formation of 5,10,15,20-tetra( m -hydroxyphenyl) porphyrin ( m -THPP), hydroxy m -THPP and three hydroxy m -THPC isomers. Hydroxylation on the reduced pyrrole ring of m -THPC was the most prominent reaction.

Time-dependent porphyric response in mice subchronically exposed to arsenic

1 A time-course study was carried out in mice subchroni cally exposed to As III (as sodium arsenite) or As V (as sodium arsenate), via drinking water, relating the pattern of urinary porphyrin excretion to the renal and hepatic enzyme activities of porphobilinogen deaminase (PBGD), uroporphyrinogen III synthetase (URO III-S), uropor phyrinogen decarboxylase (URO-D) and coproporphyrino gen oxidase (COPRO-O), as well as to the hepatic por phyrin accumulation in the treated animals. 2 A time-dependent, wave-like porphyric response was found in mice exposed to As V, and the increases seen in total urinary porphyrins (at 3 weeks of exposure) corre sponded to an increased activity of PBGD and Uro III-S in liver. 3 Significant decreases in renal URO-D and hepatic and renal COPRO-O activities were found in treated mice; these inhibitions were more pronounced in animals exposed to As III. 4 The combination of these enzymic effects may explain the time-dependent porphyric response of mice subchroni cally exposed to As. Finally, the relative magnitudes of URO-D and COPRO-O inhibitions may determine the pat tern of porphyrin concentration observed in urine and tis sues. 5 The decrease in renal URO-D activity may help to explain the inversion in the coproporphyrin/uroporphyrin ratio previously reported in humans chronically exposed to As; however, there were differences between the uri nary porphyrin profiles found in both species. The possi ble reasons for the similarities and differences are briefly discussed.

Analysis of aryltetrahydronaphthalene lignans and their glucoside conjugates in podophyllin resin by high-performance liquid chromatography

Abstract A reversed-phase high-performance liquid chromatography (HPLC) system has been developed and optimised for the separation of aryltetrahydronaphthalene lignans of the podophyllotoxin series and their glucoside conjugates. The separations were carried out on an ODS-Hypersil column with CH3OH-water, CH3CN-water, CH3OH-ammonium acetate or CH3CN-ammonium acetate as the mobile phase. The optimised HPLC system has been applied to the analysis of lignans in podophyllin resins. The separations showed the presence of podophyllotoxin, 4′-demethylpodophyllotoxin, podophyllotoxin δ- d -glucoside and 4′-demethylpodophyllotoxin-β- d -glucoside in Podophyllum emodi resin, while in Podophyllum peltatum resin, α- and β-peltatin and their glucoside conjugates were also detected, in addition to podophyllotoxin.

Determination of 5-aminolaevulinic acid dehydratase activity in erythrocytes and porphobilinogen in urine by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatographic (MECC) method has been developed and optimised for the separation of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG). The running buffer consisted of a mixture of 20 mM sodium phosphate and 20 mM sodium borate containing 50 mM sodium dodecyl sulphate (SDS) adjusted to pH 9.5 with 1 M NaOH. The running voltage and temperature were 20-25 kV and 30 degrees C, respectively. The MECC method for the analysis of PBG is fast and simple and is useful for the screening of PBG in the urine of patients suspected to have acute intermittent porphyria (AIP), and for the confirmation of lead exposure by measuring red-cell ALA-dehydratase (ALA-D) activity with ALA as the enzyme substrate.

Peroxidase Activation of 4-Hydroxytamoxifen to Free Radicals Detected by EPR Spectroscopy

4-Hydroxytamoxifen is a major metabolite of the antiestrogenic drug tamoxifen used in the treatment of women with breast cancer. 4-Hydroxytamoxifen is broken down by a horseradish peroxidase/H2O2 system very much more rapidly than tamoxifen and causes much greater DNA damage determined by 32P-postlabelling. EPR spin trapping of 4-hydroxytamoxifen reaction products in the presence of the free radical trap 5,5-dimethyl-1-pyrroline N-oxide, together with glutathione as a hydrogen donor, resulted in the generation of a species with the characteristics of the glutathione thiyl radical (aN approximately 15.3 G, aH approximately 16.2 G). Support for the creation of thiyl radicals comes from the close to stoichiometric time dependent formation of glutathione disulfide concomitant with the loss of glutathione. Similar results were obtained using 4-hydroxytoremifene but no radical formation or glutathione loss could be detected using 3-hydroxytamoxifen (droloxifene). On-line LC-ESI MS analysis of the incubation products from 4-hydroxytamoxifen has identified three products with a protonated molecular mass of 773, consistent with the formation of dimers of 4-hydroxytamoxifen. The role that radical mechanisms have in the carcinogenic effects of tamoxifen in the endometrium or other target organs of women taking this drug remains to be established.

Preparation and separation of hydroxy derivatives of uroporphyrinogen I by high-performance liquid chromatography with electrochemical detection

The preparation and high-performance liquid chromatography (HPLC) separation of meso-hydroxyuroporphyrinogen I, hydroxyacetic acid uroporphyrinogen I and beta-hydroxypropionic acid uroporphyrinogen I is described. meso-Hydroxyuroporphyrin I, hydroxyacetic acid uroporphyrin I and beta-hydroxypropionic acid uroporphyrin I were isolated from the urine of a patient with congenital erythropoietic porphyria. The porphyrins were reduced to the corresponding porphyrinogens with 3% (w/w) Na/Hg amalgam. The hydroxy porphyrinogens were separated on a Hypersil ODS column with 4% (v/v) acetonitrile in 1 M ammonium acetate buffer, pH 5.16, containing EDTA (0.27 mM) as the mobile phase, and detected electrochemically. Reduction of meso-hydroxyuroporphyrin I and hydroxyacetic acid uroporphyrin I, followed by HPLC analysis, showed that, in addition to the expected formation of meso-hydroxyuroporphyrinogen I and hydroxyacetic uroporphyrinogen I, respectively, uroporphyrinogen I was also produced. Reduction of beta-hydroxypropionic acid uroporphyrin I, however, gave beta-hydroxypropionic acid uroporphyrinogen I, acrylic acid uroporphyrinogen I and uroporphyrinogen I as the products. The peaks were identified by conversion into the porphyrin methyl esters and analysed by liquid secondary-ion mass spectrometry.