Spectrophotometric Assay for Processes Involving Changes in Hydrogen Ion Concentration in Aqueous Solution

Penketh, Philip G.; Shyam, Krishnamurthy; Patton, Curtis L.; Sartorelli, Alan C.

doi:10.1006/abio.1996.0248

Cited by 11 publications

(11 citation statements)

References 4 publications

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“…3,13 We utilized a simple colorimetric assay that relies upon the measurement of the relatively linear change in absorption at 560 nm of phenol red that occurs in proportion to the generation/addition of small quantities of hydrogen ions when a weakly buffered solution of this pH indicator is subjected to incremental acidification over a narrow pH range (ΔpH < 0.1 unit) close to the p K a values of the buffer/indicator components. 34 Using a 20 μg/mL solution of phenol red in 10 mM Tris-HCl/10 mM GSH buffer, the absorbance at 560 nm was followed at an initial pH value of 7.4 at 37 °C upon addition of 100 μM 90CE (10 μL/mL of 10 mM 90CE in DMSO), in the presence and absence of GSTA1 0.5 mg/mL. The assay mixtures were sealed with parafilm in 1 mL cuvettes to minimize changes in pH due to CO 2 exchange, and they were brought to the appropriate temperature prior to the addition of agent by injection through the parafilm and rapid mixing.…”

Section: Methodsmentioning

confidence: 99%

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Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine

Penketh

Patridge

Shyam

et al. 2014

Chem. Res. Toxicol.

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Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 ∼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O6-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N3-cytosinyl),-2-(N1-guaninyl)ethane DNA interstrand cross-links via N1,O6-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.

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Section: Methodsmentioning

confidence: 99%

“…Some of these components (Tris in particular) exhibit significant temperature dependent shifts in their p K a values; therefore, the reaction mixture pH must be set at the experimental temperature. 13,34 …”

Section: Methodsmentioning

confidence: 99%

Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine

Penketh

Patridge

Shyam

et al. 2014

Chem. Res. Toxicol.

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show abstract

“…We utilized a simple colorimetric assay that relies upon the measurement of the relatively linear change in absorption at 560 nm of phenol red that occurs in proportion to the generation/addition of small quantities of hydrogen ions when a weakly buffered solution of this pH indicator is subjected to incremental acidification over a narrow pH range (ΔpH < 0.1 unit) close to the p K a values of the buffer/indicator components. 28 Using a 20 μg/mL solution of phenol red in 2 mM Tris-HCl buffer, the absorbance at 560 nm was followed at an initial pH value of 7.4 at 20 and 37 °C upon the addition of 50 μM 90CE (5 μL/mL of 10 mM 90CE in DMSO). The assay mixtures were sealed with parafilm in 1 mL cuvettes to minimize changes in pH due to CO 2 exchange and brought to the appropriate temperature prior to the addition of agent via injection through the parafilm and rapid mixing.…”

Section: Methodsmentioning

confidence: 99%

Influence of Phosphate and Phosphoesters on the Decomposition Pathway of 1,2-Bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the Active Anticancer Moiety Generated by Laromustine, KS119, and KS119W

Penketh

Shyam

Zhu

et al. 2014

Chem. Res. Toxicol.

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Prodrugs of the short-lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine (KS90) are potentially useful anticancer agents. This class of agents frequently yields higher ratios of therapeutically active oxophilic electrophiles responsible for DNA O6-guanine alkylations to other electrophiles with lower therapeutic relevance than the nitrosoureas. This results in improved selectivity toward tumors with diminished levels of O6-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O6-alkylguanine repair. The formation of O6-(2-chloroethyl)guanine, which leads to the formation of a DNA–DNA interstrand cross-link, accounts for the bulk of the anticancer activity of 90CE prodrugs. Herein, we describe a new decomposition pathway that is available to 90CE but not to its methylating counterpart. This pathway appears to be subject to general/acid base catalysis with phosphate (Pi), phosphomonoesters, and phosphodiesters, being particularly effective. This pathway does not yield a chloroethylating species and results in a major change in nucleophile preference since thiophilic rather than oxophilic electrophiles are produced. Thus, a Pi concentration dependent decrease in DNA–DNA interstand cross-link formation was observed. Changes in 90CE decomposition products but not alkylation kinetics occurred in the presence of Pi since the prebranch point elimination of the N-1 methanesulfinate moiety remained the rate-limiting step. The Pi catalyzed route is expected to dominate at Pi and phosphoester concentrations totaling >25–35 mM. In view of the abundance of Pi and phosphoesters in cells, this pathway may have important effects on agent toxicity, tumor selectivity, and resistance to prodrugs of 90CE. Furthermore, it may be possible to design analogues that diminish this thiophile-generating pathway, which is likely superfluous at best and potentially detrimental to the targeting of hypoxic regions where Pi concentrations can be significantly elevated.

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“…Decomposition Kinetics of NaBH 4 -The decomposition kinetics of NaBH 4 in 20 mM Tris-HCl buffer containing 1 mM EDTA and 20 g/ml phenol red at pH 7.4 were determined by following the change in absorbance of phenol red at 560 nm, which varies linearly with very small molar changes in the consumption or generation of hydrogen ions (21). The measurements were based upon the alkalinization of the medium from the consumption of 1 mol of protons/mol of NaBH 4 during its decomposition at pH values close to neutrality, as represented in the following reaction.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

Penketh¹,

Hodnick²,

Belcourt³

et al. 2001

Journal of Biological Chemistry

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Mitomycin C requires reductive activation to crosslink DNA and express anticancer activity. Reduction of mitomycin C (40 M) by sodium borohydride (200 M) in 20 mM Tris-HCl, 1 mM EDTA at 37°C, pH 7.4, gives a 50 -60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t1 ⁄2 of ϳ15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA crosslinking, regenerated 10 -70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/ hypoxic toxicity differentials and resistance can occur.The mitomycin antibiotics, produced by various Streptomyces, give rise to electrophiles capable of cross-linking DNA following either one-electron or two-electron reduction by enzymatic or chemical systems (1). The one-electron reduction product, the mitomycin C semiquinone radical anion (MC . ), 1 reacts with molecular oxygen (itself a stable diradical) at close to the diffusion controlled rate (10 9 to 10 10 M Ϫ1 s Ϫ1 ) to give the parent mitomycin C (MC) quinone and superoxide (O 2 . ) (2).Since very few cross-links are required to give rise to a lethal event (3), the regeneration of MC and the production of a superoxide anion, a species of low toxicity relative to a DNA cross-link, represents a detoxification step. Under physiological oxygen concentrations, the half-life of MC . would be expected to be less than 0.1 ms. Therefore, in the presence of physiological concentrations of oxygen, only an extremely small proportion of the MC . produced could be involved in the direct alkylation of biomolecules, and the yields of DNA cross-links via this pathway would be negligible. The two-electron reduced species, mitomycin C hydroquinone (MCH 2 ), does not react rapidly with oxygen and, therefore, cross-links DNA in a manner largely independent of the concentration of oxygen. Thus, the degree of initial DNA damage by MC under aerobic conditions almost exclusively depends upon a two-electron reduction to MCH 2 . Under very low oxygen concentrations, greater cellular damage would be expected, reflecting the rate of production of both MCH 2 and MC . . There is also evidence that most of the damage resulting from the production of MC . under hypoxic conditions is due to disproportionation (4) or further reductio...

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Spectrophotometric Assay for Processes Involving Changes in Hydrogen Ion Concentration in Aqueous Solution

Cited by 11 publications

References 4 publications

Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine

Influence of Glutathione and Glutathione S-transferases on DNA Interstrand Cross-Link Formation by 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the Active Anticancer Moiety Generated by Laromustine

Influence of Phosphate and Phosphoesters on the Decomposition Pathway of 1,2-Bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the Active Anticancer Moiety Generated by Laromustine, KS119, and KS119W

Inhibition of DNA Cross-linking by Mitomycin C by Peroxidase-mediated Oxidation of Mitomycin C Hydroquinone

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