A dosimetric analysis comparing electron beam with the MammoSite brachytherapy applicator for intact breast boost.

INTRODUCTION: Electron beam radiation is the modality most often used to deliver an operative bed boost to breast cancer patients after completing whole breast radiation. However, electrons can potentially provide inadequate coverage. The MammoSite breast brachytherapy applicator may provide dosimetric advantages in the delivery of an operative bed boost and its role in this setting is not yet defined. MATERIALS AND METHODS: The study population consisted of 15 patients with early stage breast cancer treated with partial breast irradiation (PBI) using the MammoSite device. For each patient, a theoretical boost plan using electrons and a second theoretical boost plan using the MammoSite applicator were created. To assess the adequacy of each boost plan, the PTV V90, PTV V95, and PTV V100 were calculated. To assess dose to normal tissues, the ipsilateral breast V50, ipsilateral lung V30, and heart V20 were calculated. RESULTS: The mean PTV V100 for the MammoSite boost was 95.5%, compared to 77.4% for the electron boost (p<0.001). The mean PTV V95 was 97.8%, compared to 93.3% for the electron boost (p=0.02). The mean PTV V90, mean breast V50, mean lung V30, and mean heart V20 were not statistically different for MammoSite compared to electrons. CONCLUSIONS: A tumor bed boost using the MammoSite breast brachytherapy applicator provides superior target coverage and delivers similar doses to the ipsilateral breast and lung compared to a boost delivered with electrons. More investigation into the role of balloon brachytherapy in the delivery of a breast boost is warranted.

Novel fatty acid esters of p-coumaryl alcohol in epicuticular wax of apple fruit.

Hexane extracts of epicuticular wax from cv. Gala apples were noted to have an unusual, broad absorbance maximum at approximately 258 nm, which led us to isolate and identify the primary UV-absorbing compounds. Column and thin-layer chromatography yielded a fraction that gave a series of paired, 260-nm-absorbing peaks on C(18) HPLC. These were shown to be a family of phenolic fatty acid esters, for which retention times increased with increasing fatty acid chain length, and paired peaks were esters of two related phenolics with the same fatty acid moiety. Alkaline hydrolysis of the esters released two water-soluble phenolics separable by C(18) HPLC. Electrospray ionization mass spectrometry gave a molecular mass of 150 for both, and (1)H NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol. Alkaline cleavage of the fatty acid esters in the presence of methanol or ethanol resulted in partial derivatization of E-p-coumaryl alcohol to the corresponding gamma-O-methyl or O-ethyl ether. Gradient HMQC NMR of the HPLC-purified stearate ester of E-p-coumaryl alcohol indicated that fatty acid esterification occurs at the gamma-OH rather than at the 4-OH on the phenyl ring. This is the first report of fatty acid esters of monolignols as a natural plant product.

Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids.

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.

p-Cresol sulfate is the dominant component of urinary myelin basic protein like material.

Multiple sclerosis (MS) is clinically heterogeneous and has an uncertain natural history. A high priority for more effective treatment of MS is an objective and feasible laboratory test for predicting the disease's course and response to treatments. Urinary myelin basic protein (MBP)-like material (MBPLM), so designated because it is immunoreactive as a cryptic epitope in peptide 83-89 of the human MBP molecule of 170 amino acids, is present in normal adults, remains normal in relapsing-remitting, but increases in progressive MS. In the present investigation, MBPLM was purified from urine and characterized. p-Cresol sulfate is the major component of urinary MBPLM. This conclusion is based on the following: (1) MBPLM and p-cresol sulfate both have a mass of 187 on negative scans by electrospray ionization mass spectrometry, the same fragments on tandem mass spectrometry of 80 (SO(-)(3)) and 107 (methylphenol), and similar profiles on multiple reaction monitoring; (2) (1)H and (13)C nuclear magnetic resonance spectroscopy revealed identical spectra for MBPLM and p-cresol sulfate; (3) purified p-cresol sulfate reacted in parallel with MBP peptide 83-89 in the same radioimmunoassay for MBPLM; and (4) p-cresol sulfate has the same behavior on preparative HPLC columns as urinary MBPLM. The unexpected immunochemical degeneracy permitting a cross-reaction between p-cresol sulfate and a peptide of an encephalitogenic myelin protein is postulated to be based on shared conformational features. The mechanisms by which urinary p-cresol sulfate, possibly derived from tyrosine-SO(4), reflects progressive worsening that is disabling in MS are unknown.

Synthesis of the prototype DNA-protein cross-link, 1-(guan-1-yl)-2-(cysteine-S-yl)ethane, and its role in the reactions of the haloethylnitrosoureas.

The haloethylnitrosoureas form a cytotoxic DNA cross-link in a series of reactions which involves initial alkylation of the O6 position of guanine and rearrangement to the intermediate, 1,O6-ethanoguanine; 1,O6-ethanoguanine then reacts with a neighboring cytosine base. O6-Alkylguanine-DNA alkyltransferase can interrupt this process after the initial alkylation step by removing the alkyl group from the O6 position of guanine. Recent evidence suggests that the O6-alkylguanine-DNA alkyltransferase also recognizes 1,O6-ethanoguanine as a substrate, becoming bound to DNA when it interacts with that intermediate. It has also been shown that glutathione becomes bound to haloethylnitrosourea-treated DNA, apparently through chemical interaction with 1,O6-ethanoguanine. Since both of these reactions involve the thiol group of cysteine, we have examined the reaction of cysteine with 1,O6-ethanoguanine, characterizing the prototype DNA-protein cross-link, 1-(3-cytosinyl),2-(1-guanyl)ethane, which is formed in this reaction. These results establish a competitive reaction with 1,O6-ethanoguanine as a likely route to protein-DNA cross-linking.

Cardiovascular risk factors in children: a Bloomsday research report.

Cardiovascular risk factors identified in major studies of children include a family history of heart disease, smoking, blood pressure, dietary factors, exercise or fitness level, and stress. The purpose of this descriptive study was to identify differences in children's physical measurements with regard to six cardiovascular risk factors. A descriptive design using three questionnaires and physical measurements was used. The questionnaires included the Bloomsday Cardiovascular Fitness Questionnaire, the Coronary Risk Profile, and the Diet Habit Survey. Physical measurements included blood pressure readings, weight, total serum cholesterol, and high-density lipoprotein levels. A convenience sample of 78, predominantly Caucasian children between the ages of 7 and 18 years (M = 13.13, SD = 2.79) participated. Results indicated that differences in physical measurements were significantly for children with respect to the cardiovascular risk factors, including dietary factors, blood pressure, exercise, and stress. Nursing implications for thorough assessment and intervention through education are discussed.

Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II.

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three. As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine. In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.

Formation of N2,3-ethanoguanine in DNA after in vitro treatment with the therapeutic agent, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea.

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.

Mechanism of action of the nitrosoureas--IV. Synthesis of the 2-haloethylnitrosourea-induced DNA cross-link 1-(3-cytosinyl),2-(1-guanyl)ethane.

The 2-haloethylnitrosoureas have been shown to form the cross-linked structure 1-(3-cytosinyl),2-(1-guanyl)ethane in DNA. This cross-link has now been synthesized by the reaction of O6-(2-fluoroethyl)guanosine with deoxycytidine in dimethyl sulfoxide followed by removal of the sugars by acid hydrolysis. This synthetic route supports the mechanism for cross-link formation in DNA that involves an initial attack on the O6-position of guanine, followed by a rearrangement and subsequent reaction with cytosine. It also provides a practical route to the synthesis of 1-(3-cytosinyl),2-(1-guanyl)ethane for studies involving formation of this cross-link in DNA.

Phosphotriester formation by the haloethylnitrosoureas and repair of these lesions by E. coli BS21 extracts.

The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers. Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation. A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA. An extract of E. coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.

Reaction of reduced metronidazole with guanosine to form an unstable adduct.

Metronidazole, which is known to react with DNA under certain conditions, forms an adduct with guanosine in the presence of the reducing agent, sodium dithionite. This product has been purified by HPLC, characterized by UV and fast atom bombardment mass spectrometry, and tentatively identified as 1-(2-hydroxyethyl)-2-methyl-5-(N2-guanosinylamino)imidazole. This adduct is very unstable and decomposes to a variety of products including guanosine. Formation of this and similar DNA adducts from metronidazole in vivo, and the decomposition of these products, are probably both strongly dependent on details of intracellular metabolism.

Synthesis and characterization of O6-(2-chloroethyl)guanine: a putative intermediate in the cytotoxic reaction of chloroethylnitrosoureas with DNA.

The chloroethylnitrosoureas are useful antitumor agents which evidently exert a significant part of their cytotoxic action through the formation of a unique 1-(3-deoxycytidyl), 2-(1-deoxyguanosinyl)ethane cross-link in DNA. It has been suggested that this cross-link is formed from O6-(2-chloro-ethyl)guanine, an initial product of DNA alkylation by the chloroethylnitrosoureas; however, O6-(2-chloroethyl)guanine has never been described. We have synthesized this derivative from the reaction of thionyl chloride with O6-(2-hydroxyethyl)guanine, and have found that it decomposes to 1-(2-hydroxyethyl)guanine through an intermediate, presumably 1,O6-ethanoguanine. Its half life at 37 degrees and pH 7.4 is 17.8 min.

Spectroscopic detection of iminocyclophosphamide and its possible role in cyclophosphamide metabolism.

Iminocyclophosphamide (4) has been identified by 1H NMR as a product from base treatment of 4-alkylthio-substituted cyclophosphamide derivatives, viz., cis-4-(propylthio)cyclophosphamide (cis-7). A maximum concentration of approximately 12% of total product was observed by treating cis-7 with ethyl propiolate and NaH or deuteriated dimsyl anion in anhydrous Me2SO-d6. Treatment of cis-7 with base alone established a rapid cis-/trans-7 equilibrium via the imine intermediate 4. Base-catalyzed expulsion of 1-propanethiol (8) from cis-7 and thiol trapping afforded formation of 4, which subsequently underwent elimination to the relatively more stable conjugated (vinylimino)-phosphamide (9). Iminocyclophosphamide (4) was also identified by fast atom bombardment mass spectrometry as a product generated upon analysis of cyclophosphamide derivatives substituted in the 4-position of the oxazaphosphorine ring with various leaving groups.

Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) with guanosine: evidence for a new mechanism of DNA modification.

When guanosine reacts with 1,3-bis(2-chloro-2,2-dideuteroethyl)-1-nitrosourea in a mixture of pH 7.1 buffer and DMSO, the 7-chloroethylguanosine which is isolated contains two deuterium atoms located next to the guanine ring and beta to the chlorine atom as shown by electron impact mass spectrometry. It is proposed that initial attack by DNA bases occurs on the number 2 carbon of the haloethylnitrosourea with displacement of the chloride ion. In accordance with this proposed mechanism, 7-bromoethylguanosine is isolated as a major product when BCNU is reacted with guanosine in the presence of high concentrations of KBr. These results suggest that the antitumor activity of various haloethylating antitumor agents may be determined by structural changes which affect their mechanisms of reaction with DNA.

Isolation, synthesis, and antitumor evaluation of spirohydantoin aziridine, a mutagenic metabolite of spirohydantoin mustard.

Spirohydantoin mustard (SHM), a central nervous system directed nitrogen mustard with anticancer activity, was metabolized in the presence of mouse liver postmitochondrial supernatant (9000g fraction) to a nonpolar alkylating metabolite. The metabolite was isolated by thin-layer chromatography of chloroform or ethyl acetate extracts of incubation mixtures, and its structure was established by mass spectral analysis, synthesis, and cochromatography. The metabolite, spirohydantoin aziridine, was mutagenic for Salmonella typhimurium TA1535 in the Ames assay but inactive as an antitumor agent against P388 leukemia in vivo.

Formation of O6-ethylthioethyldeoxyguanosine from the reaction of chloroethyl ethyl sulfide with deoxyguanosine.

O6-Ethylthioethyldeoxyguanosine has been synthesized from 6-chloro-3',5'-di-O-acetyldeoxyguanosine and characterized by UV, fluorescence, and mass spectrometry. High-pressure liquid chromatography studies have shown that this modified nucleoside is formed when the one-armed sulfur mustard, chloroethyl ethyl sulfide, reacts with deoxyguanosine. This result supports the hypothesis that the mutagenic effects of the sulfur mustards are caused in part by substitution of the O6-position of deoxyguanosine.

Synthesis and properties of some 13-cis- and all-trans-retinamides.

Several 13-cis-retinamides were synthesized from 13-cis-retinoic acid via either 13-cis-retinoyl chloride or 13-cis-1-retinoylimidazole. All-trans-retinoylglycine was prepared from all-trans-retinoyl chloride and ethyl glycinate. Detailed procedures were developed for the preparation of other all-trans-retinamides on a large scale for studies of the chemoprevention of cancer.

Mechanism of action of the nitrosoureas--V. Formation of O6-(2-fluoroethyl)guanine and its probable role in the crosslinking of deoxyribonucleic acid.

DNA which has been exposed to 2-haloethylnitrosoureas has been shown to contain the chemical crosslink 1-(N3-deoxycytidyl), 2-(N1-deoxyguanosinyl)-ethane [W. P. Tong, M. C. Kirk and D. B. Ludlum, Cancer Res. 43, 3102 (1982)]. We have hypothesized that this structure is formed by an initial attack of a 2-haloethyl group on the 6 position of guanine followed by an intramolecular rearrangement and secondary crosslinking reaction with cytosine. We have now shown that DNA which had been reacted with N-(2-fluoroethyl)-N'-cyclohexyl-N-nitrosourea contained O6-(2-fluoroethyl)guanine and have thus demonstrated that the first step in the proposed mechanism occurs. Furthermore, O6-(2-fluoroethyl)guanosine hydrolyzed to N1-(2-hydroxyethyl)guanosine, showing that the necessary intramolecular rearrangement occurs. These two observations greatly strengthen the proposed route to DNA crosslinking by the 2-haloethylnitrosoureas.

Refined carbohydrate as a contributing factor in reactive hypoglycemia.

To assess the effects of diet on symptoms in patients with idiopathic reactive hypoglycemia, we obtained by dietary recall histories the dietary composition from eight of these patients. The data obtained represented the dietary content of each individual before and after instruction on a diet containing 100 gm of carbohydrate daily. Neither the percentage of carbohydrate or fat nor the total grams of protein or fat changed significantly after dietary instruction. In contrast, the percentage of refined carbohydrate decreased (P = .003) and the percentage of protein increased (P = .003). The total grams of refined carbohydrate and total carbohydrate both decreased significantly (P = .01 and .05, respectively), but the fall in refined carbohydrate (from 147 to 20 gm) accounted for the entire fall in total dietary carbohydrate. All values except those of refined carbohydrate were within 95% confidence limits of the average diet in Western culture. Despite continuing intake of a normal quantity of total carbohydrate (mean, 221 gm), all patients had symptomatic improvement. These data suggest that reducing the amount of dietary refined carbohydrate may be important in the treatment of idiopathic reactive hypoglycemia, and that quantitative reduction in total carbohydrate may be of lesser importance.

Formation of the cross-link 1-[N3-deoxycytidyl),2-[N1-deoxyguanosinyl]ethane in DNA treated with N,N'-bis(2-chloroethyl)-N-nitrosourea.

The cross-linked dinucleoside, 1-[N3-deoxycytidyl],2-[N1-deoxyguanosinyl]ethane, has been isolated from DNA which has been exposed to N,N'-bis(2-chloroethyl)-N-nitrosourea. It is probable that this structure is responsible for the interstrand cross-linking observed previously by physical methods. The modification is unique in that it cross-links DNA through two base-pairing positions and probably arises through the transfer of a chloroethyl group to one of the bases followed by a second reaction of this group with the other strand of DNA. Initial attack could be at the N3 position of deoxycytidine, the N1 position of deoxyguanosine, or possibly the O6 position of deoxyguanosine. Attack at the O6 position of deoxyguanosine would require an internal cyclization with the N1 position of deoxyguanosine before secondary reaction with the N3 position of deoxycytidine but would explain resistance to N,N'-bis(2-chloroethyl)-N-nitrosourea in cells capable of removing substituents on the O6 position of guanine.