*2.6. Lifestyle Exposures*

Another reason for spermatozoa to become oxidatively stressed relates to environmental and lifestyle exposures that either directly promote ROS generation or suppress levels of intrinsic antioxidant protection. A classic example is smoking. If men smoke heavily, their entire physiology is oxidatively stressed, as reflected by lower levels of antioxidants, such as ascorbate, in seminal plasma as well as enhanced levels of ROS generation by the spermatozoa. A 48% increase in seminal leukocyte concentration in male smokers also contributes to the level of redox stress experienced by the spermatozoa [85]. A particular pathological feature of cigarette smoking is that it generates a significant increase in oxidative DNA damage in spermatozoa as reflected by elevated levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) [86]. This increase in oxidative DNA damage may be attributable to both the above-mentioned depletion of antioxidant protection as well as the suppressive impact of cadmium (a critical constituent of cigarette smoke) on OGG-1 (8-oxoguanine-DNA glycosylase-1; the first enzyme in the base excision repair pathway responsible for removing 8-OHdG adducts from the genome) during spermatogenesis [87]. The high levels of 8-OHdG present in the spermatozoa of male smokers has, in turn, been associated with the increased childhood cancer rates observed in the offspring of male smokers [88,89]. Interestingly, we have identified a region of chromosome 15 (15q13–15q14) as a particular hot spot for oxidative DNA damage in human spermatozoa [90] and this is the very region of the genome that has recently been identified as contributing to the aetiology of acute lymphoblastic leukemia [91], one of the childhood cancers that we know to be associated with paternal smoking [92].

Obesity is another lifestyle factor responsible for inducing a state of oxidative stress in spermatozoa. This condition is associated with a generalized pro-inflammatory state associated with systemic oxidative stress, antioxidant depletion and oxidative sperm DNA damage [93]. Fortunately, this situation can be reversed, at least in mice, by the concomitant administration of micronutrients (zinc, selenium, lycopene, vitamins E and C, folic acid, and green tea extract) to counter the oxidative stress [94].

Di fferent frequencies of electromagnetic radiation have also been suggested to induce oxidative stress in the male germ line. The impact of radiofrequency electromagnetic radiation (RF-EMR) has recently been reviewed and supports the general consensus that, like obesity, this form of radiation can induce ROS generation, reduce antioxidant protection and increase sperm DNA damage [95]. Moreover, a 2-step mechanism has been proposed to explain this phenomenon; in the first step, RF-EMR induces displacement of electrons from the electron transport chain in the inner mitochondrial membrane. These leaked electrons are immediately taken up by the universal electron acceptor, oxygen, to generate superoxide anion. In the second step, the latter dismutates into the powerful oxidant, hydrogen peroxide, which then initiates a lipid peroxidation cascade culminating in the generation of cytotoxic electrophilic aldehydes such as 4-hydroxynonenal (4-HNE). This aldehyde then immediately seeks out the nucleophilic centers of proteins in the immediate vicinity, alkylating the latter at the vulnerable amino acids, cysteine, lysine and histidine. The alkylation of proteins in the electron transport chain, such as succinic acid dehydrogenase, further disrupts the flow of electrons within the inner mitochondrial membrane leading to ye<sup>t</sup> more ROS generation and exacerbating the state of oxidative stress, ultimately compromising both the functional competence of the spermatozoa and the integrity of their DNA [96,97]. The clinical significance of these findings can be found in several epidemiological studies demonstrating significant links between mobile phone usage and semen quality [95].

Importantly, it has also been clearly demonstrated that these RF-EMR e ffects are not mediated by increased heat. This is critical because raised intratesticular temperature is another means by which EMR can influence sperm quality. Such thermo-sensitivity is reflected in the location of the testes in a scrotal sac resulting in an intratesticular temperature of 34 ◦C–35 ◦C, 2–3 ◦C below the core body temperature of 37 ◦C. If intratesticular temperatures are raised by, for example, experimental cryptorchidism, then there is a rapid cessation of germ cell di fferentiation triggered by a sudden wave of Fas-mediated apoptosis and autophagy in pachytene spermatocytes and spermatids via mechanisms that can be reversed by the administration of antioxidants [98,99]. Similarly, the loss of fertility induced by warming the scrotal mouse testes to 42 ◦C for 30 min can be completely reversed by the administration of antioxidants [35]. Another situation in which raised intratesticular temperature is evident is varicocele. This condition is also known to be associated with a loss of sperm function and DNA integrity as a consequence of oxidative stress [100,101]. Surgical ligation of the left internal spermatic vein to correct the varicosity has been shown to reduce oxidative stress parameters and to improve sperm quality and the concomitant administration of antioxidants has been shown to facilitate this process [102,103]. As we enter an era of elevated ambient temperatures associated with global climate change, we might anticipate an increased incidence of male infertility, as well as an elevated risk of a paternally-induced mutations in the o ffspring, as a consequence of oxidative DNA damage to the spermatozoa. It is already known that exposure to elevated summer temperatures suppresses human seminal quality and that oxidative stress is a major mediator of this change [104,105]. Such exposure will be of particular significance to livestock species where exposure to elevated ambient temperatures might not only suppress fertility but also disrupt the true breeding of selected genetic traits [106].
