Ice slurry on outdoor running performance in heat.

The efficacy of ingestion of ice slurry on actual outdoor endurance performance is unknown. This study aimed to investigate ice slurry ingestion as a cooling intervention before a 10 km outdoor running time-trial. Twelve participants ingested 8 g · kg (- 1) of either ice slurry ( - 1.4°C; ICE) or ambient temperature drink (30.9°C; CON) and performed a 15-min warm-up prior to a 10 km outdoor running time-trial (Wet Bulb Globe Temperature: 28.2 ± 0.8°C). Mean performance time was faster with ICE (2 715 ± 396 s) than CON (2 730 ± 385 s; P=0.023). Gastrointestinal temperature (Tgi) reduced by 0.5 ± 0.2°C after ICE ingestion compared with 0.1 ± 0.1°C (P<0.001) with CON. During the run, the rate of rise in Tgi was greater (P=0.01) with ICE than with CON for the first 15 min. At the end of time-trial, Tgi was higher with ICE (40.2 ± 0.6°C) than CON (39.8 ± 0.4°C, P=0.005). Ratings of thermal sensation were lower during the cooling phase and for the first kilometre of the run ( - 1.2 ± 0.8; P<0.001). Although ingestion of ice slurry resulted in a transient increase in heat strain following a warm up routine, it is a practical and effective pre-competition cooling manoeuvre to improve performance in warm and humid environments.

Screening for xenobiotic electrophilic metabolites using pulsed ultrafiltration-mass spectrometry.

A pulsed ultrafiltration-mass spectrometric screening assay has been developed to generate and identify electrophilic metabolites of xenobiotic compounds formed by hepatic cytochrome P450 enzymes. This assay would be suitable for the early identification of potentially toxic compounds during the initial phase of drug development. Rat liver microsomes were trapped by an ultrafiltration membrane in a stirred flow-through chamber, and substrates for microsomal cytochrome P450 including hydroxychavicol, 3-methylindole, cyproheptadine and 2-tert-butyl-4,6-dimethylphenol were flow-injected individually through the chamber along with the cofactors, NADPH and glutathione. Metabolites and glutathione conjugates were detected on-line using electrospray mass spectrometry. Alternatively, the ultrafiltrate was concentrated on a reversed phase HPLC column and analyzed using electrospray LC-MS or LC-MS-MS to separate and characterize isomeric metabolites and metabolites present at low concentration. Enzymatic activation of each xenobiotic substrate produced highly electrophilic metabolites such as quinones, quinone methides and imine methides that reacted with glutathione on-line to produce glutathione conjugates which were detected by using electrospray mass spectrometry. Although epoxides such as cyproheptadine epoxide were generated, it is likely that these compounds were insufficiently reactive to form glutathione conjugates in the absence of cytosolic glutathione S-transferases. Pulsed ultrafiltration-electrospray mass spectrometry offers an efficient method for in vitro formation and mass spectrometric characterization of activated microsomal drug metabolites and is suitable for use during the drug discovery process for the early identification and screening out of potentially toxic lead compounds.

Bioactivation of tamoxifen to metabolite E quinone methide: reaction with glutathione and DNA.

Despite the beneficial effects of tamoxifen in the treatment and prevention of breast cancer, long-term usage of this popular antiestrogen has been linked to an increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. Alternatively, tamoxifen could undergo O-dealkylation to give cis/trans-1,2-diphenyl-1-(4-hydroxyphenyl)-but-1-ene, which is commonly known as metabolite E. Because of its structural similarity to 4-hydroxytamoxifen, metabolite E could also be biotransformed to a quinone methide, which has the potential to alkylate DNA and may contribute to the genotoxic effects of tamoxifen. To further probe the chemical reactivity/toxicity of such an electrophilic species, we have prepared metabolite E quinone methide chemically and enzymatically and examined its reactivity with glutathione (GSH) and DNA. Like 4-hydroxytamoxifen quinone methide, metabolite E quinone methide is quite stable; its half-life under physiological conditions is around 4 h, and its half-life in the presence of GSH is approximately 4 min. However, unlike the unstable GSH adducts of 4-hydroxytamoxifen quinone methide, metabolite E GSH adducts are stable enough to be isolated and characterized by NMR and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Reaction of metabolite E quinone methide with DNA generated exclusively deoxyguanosine adducts, which were characterized by LC/MS/MS. These data suggest that metabolite E has the potential to cause cytotoxicity/genotoxicity through the formation of a quinone methide.

4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides.

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone methide formation may not make a significant contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.

Oxo substituents markedly alter the phase II metabolism of alpha-hydroxybutenylbenzenes: models probing the bioactivation mechanisms of tamoxifen.

The P450-catalyzed hydroxylation of tamoxifen to give alpha-hydroxytamoxifen [(E)-4-{4-[2-(dimethylamino)ethoxy]phenyl}-3,4-diphenyl-3-buten-2- ol] and subsequent formation of reactive sulfate esters which alkylate DNA has been proposed to be a potential carcinogenic pathway for tamoxifen. In the present study, the ability of alpha-hydroxytamoxifen analogs to form GSH and sulfate conjugates was investigated in order to understand the structural features influencing reactivity. The para oxo analogs 1 [1-(4-methoxyphenyl)-3-hydroxy-1-butene], 2 [1-(4-hydroxyphenyl)-3-hydroxy-1-butene], and 4 [1-(4-hydroxyphenyl)-1-phenyl-3-hydroxy-1-butene] reacted with GSH instantaneously under strong acidic conditions to yield GSH conjugates in greater than 90% yields. Interestingly, the meta phenolic analogs 3 [1-(3-hydroxyphenyl)-3-hydroxy-1-butene] and 5 [1-(3-hydroxyphenyl)-1-phenyl-3-hydroxy-1-butene] did not react with GSH to any significant extent under similar conditions. Characterization of the GSH conjugates with 1H-NMR, electrospray mass spectrometry, and UV showed that all of the conjugates resulted from attack of GSH at the alpha-position of the substrates with displacement of the hydroxyl group. The formation of a single pair of diastereomeric conjugates strongly supported adduct formation to proceed through a direct S(N)2 displacement mechanism and not through a quinone methide (4-alkyl-2,5-cyclohexadien-1-one) intermediate. At physiological pH and temperature only the para hydroxy analogs 2 and 4 gave GSH conjugates, a reaction which seems to be catalyzed by isoforms of glutathione S-transferase. Similar substituent effects were observed in the sulfotransferase-mediated formation of alpha-hydroxy sulfate esters in that only the para hydroxy analogs formed conjugates at the aliphatic hydroxyl group. Finally, the present investigation showed a remarkable difference in the reactivities of para and meta phenolic analogs of alpha-hydroxybutenylbenzenes toward GSH and sulfate conjugation reactions.

Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifen-o-quinone.

Although tamoxifen is approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, several studies in animal models have shown that tamoxifen is heptocarcinogenic, and in humans, tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen carcinogenesis could involve metabolism of tamoxifen to 3,4-dihydroxytamoxifen followed by oxidation to a highly reactive o-quinone which has the potential to alkylate and/or oxidize cellular macromolecules in vivo. In the study presented here, we synthesized the 3,4-dihydroxytamoxifen, prepared its o-quinone chemically and enzymatically, and studied the reactivity of the o-quinone with GSH and deoxynucleosides. The E (trans) and Z (cis) isomers of 3,4-dihydroxytamoxifen were synthesized using a concise synthetic pathway (four steps). This approach is based on the McMurry reaction between the key 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone and propiophenone, followed by selective removal of the methylenedioxy ring of (E, Z)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-(3, 4-methylenedioxyphenyl)-2-phenyl-1-butene with BCl(3). Oxidation of 3,4-dihydroxytamoxifen by activated silver oxide or tyrosinase gave 3,4-dihydroxytamoxifen-o-quinone as a mixture of E and Z isomers. The resulting o-quinone has a half-life of approximately 80 min under physiological conditions. Reaction of the o-quinone with GSH gave two di-GSH conjugates and three mono GSH conjugates. Incubation of 3,4-dihydroxytamoxifen with GSH in the presence of microsomal P450 gave the same GSH conjugates which were also detected in incubations with human breast cancer cells (MCF-7). Reaction of 3, 4-dihydroxytamoxifen-o-quinone with deoxynucleosides gave only thymidine and deoxyguanosine adducts; neither deoxyadenosine nor deoxycytosine adducts were detected. Preliminary studies conducted with human breast cancer cell lines showed that 3, 4-dihydroxytamoxifen exhibited cytotoxic potency similar to that of 4-hydroxytamoxifen and tamoxifen in an estrogen receptor negative (ER(-)) cell line (MDA-MB-231); however, in the ER(+) cell line (MCF-7), the catechol metabolite was about half as toxic as the other two compounds. Finally, in the presence of microsomes and GSH, 4-hydroxytamoxifen gave predominantly quinone methide GSH conjugates as reported in the previous paper in this issue [Fan, P. W., et al. (2000) Chem. Res. Toxicol. 13, XX-XX]. However, in the presence of tyrosinase and GSH, 4-hydroxytamoxifen was primarily converted to o-quinone GSH conjugates. These results suggest that the catechol metabolite of tamoxifen has the potential to cause cytotoxicity in vivo through formation of 3,4-dihydroxytamoxifen-o-quinone.