Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumors in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and in rat hepatocytes in culture. These arise primarily from its metabolite alpha-hydroxytamoxifen, and may also arise, in part, from another metabolite, 4-hydroxytamoxifen. We have prepared two model compounds for the potential reactive metabolite formed from 4-hydroxytamoxifen in rat liver. One of these was its alpha-acetoxy ester. This was much more reactive than that from tamoxifen, and could not be isolated in pure form. It reacted with DNA in the same way that alpha-acetoxytamoxifen did, to give adducts which were isolated by hydrolysis and chromatography, and identified as alkyldeoxyguanosines. The second derivative was alpha, beta-dehydro-4-hydroxytamoxifen. This also reacts with DNA in vitro, to give the same products as those from alpha-acetoxy-4-hydroxytamoxifen. Reaction probably proceeds through the same resonance-stabilized carbocation in either case. However, when primary cultures of rat hepatocytes were treated with either 4-hydroxytamoxifen, 4,alpha-dihydroxytamoxifen, or alpha, beta-dehydro-4-hydroxytamoxifen at a concentration of 10 microM, no adducts could be detected in their DNA by the 32P-postlabeling technique. Similarly, no adducts could be found in the liver DNA of female Fischer F344 rats treated orally (at 0.12 mmol kg-1) with the same substances. If 4-hydroxytamoxifen is metabolized to 4, alpha-dihydroxytamoxifen in rat liver, then either this substance is not converted to reactive esters or they are rapidly detoxified.

Lack of genotoxicity of tamoxifen in human endometrium.

The potential for the anti-breast cancer drug tamoxifen [(Z)-1-[4-[2-( dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] to induce genotoxic damage (DNA adducts) in the human endometrium was investigated in vivo and in vitro. Endometria from hysterectomy patients who were not on tamoxifen were sectioned and maintained in short-term organ culture. The cultures were treated with either solvent vehicle (DMSO), tamoxifen, alpha-hydroxytamoxifen [(E)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3- ol; the major DNA-reactive metabolite in the rat], or benzo(a)pyrene. DNA was isolated and analyzed by 32P postlabeling. Chromatography on polyethyleneimine-cellulose TLC plates revealed DNA adducts in endometria treated with alpha-hydroxytamoxifen identical to those seen previously in the rat liver. However, no adducts were seen from treatment with tamoxifen itself. The viability of the enzyme-metabolizing systems of the endometrial samples was demonstrated by the detection of expected DNA adducts induced by benzo(a)pyrene. Examination by liquid chromatography-mass spectrometry of the explant culture media from endometria treated with tamoxifen revealed the presence of the alpha-hydroxy metabolite in a dose-dependent manner, although apparently at levels insufficient to produce detectable DNA adducts. Endometrial DNA obtained from 18 patients undergoing daily treatment with 10-40 mg tamoxifen for 3 months-9 years was also analyzed. No evidence for any DNA adducts induced by tamoxifen was found in any of the patients examined. These data suggest that the genotoxic events observed with tamoxifen in the rat may not apply to the human endometrium.

Further metabolism of diol-epoxides of chrysene and dibenz[a,c]anthracene to DNA binding species as evidenced by 32P-postlabelling analysis.

Incubation of r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxide of chrysene, with rat liver microsomes in the presence of NADP+ and DNA, followed by 32P-postlabelling analysis of the DNA, revealed the presence of at least two adducts not detected when anti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone. The formation of these adducts was not blocked by the epoxide hydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One of the adducts cochromatographed with the adduct spot obtained when authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA. Evidence suggested that a second adduct could also be formed by further metabolism of anti-9-OH-chrysene-1,2-diol 3,4-oxide. In addition, evidence was obtained for the further metabolism of the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomer of a non-bay-region diol-epoxide of dibenz[a,c]anthracene to DNA binding species, but not for that of either the anti- or syn-isomers of the bay-region diol-epoxide of benzo[a]pyrene, the anti-isomers of the bay-region or a non-bay-region diol-epoxide of benz[a]anthracene, or the anti-isomer of the bay-region diol-epoxide of benzo[b]fluoranthene.

Metabolic activation of 7-ethyl- and 7-methylbenz[a]anthracene in mouse skin.

The carcinogen 7-methylbenz[a]anthracene (7-MBA) is considered to be metabolically activated via its bay-region dihydrodiol-epoxide, trans-3,4-dihydro-3,4-dihydroxy-7-methyl-benz[a]anthracene 1,2-oxide (7-MBA-3,4-diol 1,2-oxide). When tested on mouse skin, a target tissue for polycylic aromatic hydrocarbon carcinogenesis, 7-ethylbenz[a]anthracene (7-EBA) was much less active than 7-MBA, and this difference may be due to differences in the pathways by which the two compounds are metabolized and activated. In the present work, the metabolism by mouse-skin microsomes of both hydrocarbons to dihydrodiols has been examined. Both were metabolized to a similar extent with the 8,9-dihydrodiols being detected as the predominant metabolites. The 3,4-, 5,6-and 10,11-dihydrodiols of 7-MBA and the 3,4- and 10, 11-dihydrodiols of 7-EBA, were also detected. 7-MBA was found to bind covalently to microsomal protein at 10 times the level of 7-EBA. The covalent binding of benz[a]anthracene (BA), 7-EBA and 7-MBA to DNA in mouse skin following topical application was determined using the 32P-postlabelling assay. The results correlated with the relative carcinogenic activities of the compounds with 7-MBA binding at five and nine times the level of 7-EBA and BA respectively. For all three hydrocarbons, the major hydrocarbon: 32P-labelled nucleoside bisphosphate, eluted in the same area of the TLC maps, suggesting the involvement of a common type of bay-region dihydrodiol-epoxide intermediate.

Comparative studies of the metabolic activation of chrysene in rodent and human skin.

Metabolism and activation of chrysene was examined in mouse, rat and human skin using a short-term organ culture technique. Mouse skin released larger quantities of free dihydrodiols into the culture medium than either rat or human skin and greater quantities of chrysene metabolites became covalently bound to the DNA of mouse skin. The stereochemistry of the chrysene-1,2-diol that was formed by each skin type was examined using high-performance liquid chromatography (HPLC) with a chiral stationary phase to resolve the enantiomers. It was found that in each case the (-)-enantiomer predominated. When hydrolysates of DNA extracted from rodent or human skin that had been treated with 3H-labelled chrysene were chromatographed on Sephadex LH-20 columns, the elution profiles of the hydrocarbon-DNA adducts were found to vary between the species studied. Further examination using HPLC showed that some of the adducts formed in skin had the chromatographic characteristics of adducts formed when the anti-isomer of the 'bay-region' diol-epoxide of chrysene (r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene) reacted with DNA and that others had the characteristics of triol-epoxide adducts.

Continuous tissue pH measurement in critically ill adults with a miniaturised glass electrode.

A continuously reading subcutaneous pH electrode has been developed which gives relatively stable readings when applied to any well perfused part of the body. The results obtained with this electrode have been compared with the actual pH of the arterial blood measured by conventional methods. Thirty patients were studied and one hundred and sixty three observations were made. The correlation coefficient (r) between tissue pH (pHt) and arterial pH (pHa) was 0.76 (P less than 0.01). The response time of the electrode in vitro was less than one minute and, in vivo, stabilization of the recording occurred in less than three minutes. It was found that the discrepancy between pHa and pHt gave a good indication of the adequacy of tissue perfusion.

The role of a department of physiological (clinical) measurement in the UK National Health Service.

The relatively new concept of a centralized department of physiological measurement, and the ways in which such a department interacts with related hospital departments within the framework of the National Health Service, are be borne in mind when setting up such a department are outlined and, finally, discussed. A variety of structural and functional considerations which are to the author describes his own department in the light of these considerations.