Direct Observations of Scavenging Reactions of the Prehydrated Electron and OH Radicals by Femtosecond Time-Resolved Laser Spectroscopy

Abstract

Radiotherapy is the major curative therapy for carcinogesis. Identifying the effective species that induce DNA damage under ionizing radiation holds the key to improve and advance radiotherapy. In a cellular environment, most of the radiation energy is absorbed by water in the cell. Traditionally, the major radicals resulting from the radiolysis of water are thought to be the hydroxyl radical (OH) and the hydrated electron, whereas the (OH) radical is considered as the major contributor to radiation-induced DNA damage. With the birth of femtosecond time-resolved laser spectroscopy, the precursor to the hydrated electron, the so-called prehydrated electron, has been directly observed. The prehydrated electrons are the excited states of the well-known hydrated electron in nature. Very recently, it was pointed out that the prehydrated electron is a reactive species capable of causing lethal DNA double strand breaks. Thus the reductive DNA damage is proposed as a new molecular pathway for radiation-induced DNA damage. Therefore, the reaction dynamics of the prehydrated electron is of great interest to unravel the exact mechanism of radiation-induced DNA damage. In order to study the action of the prehydrated electron (epre–) in biologically relevant reactions, additional compounds need to be applied to regulate the prehydrated electrons. Such compounds are electron scavengers. In this thesis, the ultrafast electron transfer reaction of epre− with an electron scavenger potassium nitrate was first investigated using our state-of-the-art femtosecond time-resolved pump-probe laser spectroscopy (fs-TRLS). Quantitative scavenging efficiency is successfully obtained by measuring the reaction rate constant, which is determined to be kpre = (0.75 ± 0.5)×10^13 M^−1s^−1. This value is two-orders larger than the reaction rate constant of ehyd– with potassium nitrate k =9.7×10^9 M^−1s^−1, confirming the high reactivity of epre–. Moreover, to comparing effectiveness of the reductive DNA damage induced by the prehydrated electron to the oxidative DNA damage induced by OH radicals, OH radical scavengers are used to quench OH radicals, leaving the prehydrated electron as the only active species. However, no studies have ever investigated the reactions between OH radical scavengers and the prehydrated electron. Here we performed the first quantitative study on the scavenging reactions of epre– with the well-known OH radical scavengers, isopropanol and dimethyl sulfoxide (DMSO). We present the first evidence of such scavenging reactions and determine the reaction rate constants, which are measured to be k = 3.3 ± 0.5×10^11 M^−1s^−1 and 8.7 ± 0.5×10^11 M−1s−1 for isopropanol and DMSO in PBS buffer, respectively.These values are much higher than the reaction rate constants of isopropanol with OH radicals and DMSO with OH radicals (kisopropanol+OH = 2×10^9 M^−1s^−1 and kDMSO+OH = 7×10^9 M^−1s^−1). Furthermore, the OH radical is an important species produced from radiolysis of water. Knowing its reaction dynamics and kinetics can facilitate the comparison between the oxidative DNA damage induced by OH radicals and the reductive DNA damage by prehydrated electrons. By using an OH radical scavenger KSCN, we are able to directly observe the reaction dynamics of the OH radical. In addition, knowing the relative yield ratio of OH radicals and the epre– (r = [OH]/[epre–]) is necessary for the comparison of the effectiveness of epre– and OH radicals at inducing DNA damage. In our study, a quantitative analysis of the relative yield ratio r using an OH radical scavenger KSCN was obtained. The relative yield ratio is determined to be r = [OH]/[epre–] = 2.8 ± 0.4. Incorporating this value into our recent studies on reductive DNA damage, we find that in terms of single-strand breaks and double-strand breaks yields per radical, an epre– is nearly three times as effective as an OH at inducing DNA damage under irradiation. Overall, the results obtained from this thesis provide important information for future studies of epre– action in biologically relevant reactions.