The behavioral processes have long been suspected to influence many health conditions including cancer. Both progression and to a lesser extent cancer onset, have been related to chronic stress, depression, lack of social support and various other psychological factors (Reiche et al., 2004; Antoni et al., 2006). Stress exposure induces a cluster of physiological and behavioral changes that have been linked to the response of the organism to maintain the homeostasis. Moreover, stress has been shown to markedly influence incidence, growth, metastasis, and the rejection of both the chemically induced and implanted tumors (Amkraut and Solomon, 1972; Zygmunt et al., 1985). Both the exacerbation and attenuation of tumor development have, however, been reported in response to stress (Pradhan and Prabhati, 1974; Justice, 1985).

N-nitrosodiethylamine (NDEA) causes oxidative stress and cellular injury by enhancing the formation of free radicals (Ramakrishnan et al., 2006; Valko et al., 2006). The metabolic activation of NDEA by cytochrome P450 enzymes converts it to ethyl-acetoxyethyl-nitrosamine, which can be conjugated by the phase II enzymes (Subramanian et al., 2007) to a non-toxic compound. Alternatively, it can form ethyldiazonium ion that directly ethylates cellular macromolecules (Muzio et al., 1999), and is responsible for its cytotoxic, mutagenic, and carcinogenic effects (Abdel-Hamid et al., 2013) through the production of ROS (Bansal et al., 2000).

Oxidative stress, a pervasive condition induced by psychological stress, has been implicated in and recognized to be a prominent feature associated with various diseases like cancer and their progression. An imbalance in the pro-oxidant/antioxidant status indicates the enhanced production of reactive oxygen species (ROS), such as peroxides, hydroxyl, and superoxide anion radicals (Muqbil et al., 2006), which induces cellular oxidative damage through DNA strand breaks and lipid peroxidation (Batcioglu et al., 2002). Thus, DNA damage induced by oxidative stress and/or deficient DNA-repair has an etiological or prognostic role in cancer (Mussarat et al., 1996). A previous study (Ip et al., 1996) has demonstrated the effect of restraint stress on the antioxidant status and DNA damage in rats. It was found that chronic restraint stress increased lipid peroxidation, compromised antioxidant status and enhanced DNA damage (Zaidi et al., 2005; Muqbil and Banu, 2006; Muqbil et al., 2006). Other studies have confirmed that stress is associated with low concentrations of O₆ methyltransferase, which is an important DNA-repair enzyme (Glaser et al., 1985). Hence, it is important to correlate between alterations in the parameters of psychological stress with those of redox state, DNA-repair activity, and antioxidant defense so as to identify the possible relationship between chronic stress and cancer.

The chronic unpredictable stress (CUS) is a well-recognized model for inducing chronic physical and psychological stress. CUS has been used in the present study to assess the effects of various psychological factors and stressors on some biochemical parameters and DNA damage, also in combination with the known chemical carcinogen NDEA. This study may throw light on the effect of CUS on the early stages of liver cancer as evident from the measured biochemical markers, antioxidant status, and the DNA damage. This also suggests that the life changing unpredictable stresses may enhance the carcinogenic potential of environmental chemicals through compromised oxidative status and DNA damage.

Exposure to stress situation stimulates numerous pathways leading to increased production of free radicals. Several human chronic diseases including cancer have been associated with the altered oxidative stress, produced either through an increased free radical generation and/or a compromised antioxidant level in the target cells and tissues (Trush and Kensler, 1991; Rice-Evans and Burdon, 1993). The free radicals thus generated induce strand breaks and cause oxidative modification of DNA bases (Cerutti, 1985). The participation of ROS in hepatocarcinogenesis is evident as initiation with low doses of NDEA induces liver DNA-8-hydroxydeoxy-guanosine adduct formation (Nakae et al., 1997) and causes mutations, which leads to carcinogenesis. The free radicals, mostly ROS cause cellular injury. The consequences of which are often exhibited and measured as lipid peroxidation product MDA, the major end product of this reaction. Malondialdehyde directly reacts with DNA to form oxidative DNA adducts (Chaudhary et al., 1994), thereby causing DNA damage and interfering with the DNA-repair mechanism.

The present study shows that exposure of mice to CUS result in a significant increase in the levels of MDA, and a significant decrease in the antioxidant enzyme activities and the levels of GSH. These results are supported by previous studies in which CUS showed a disturbed oxidant-antioxidant balance (Zhang et al., 2009). The significant increase of MDA in NDEA-administered mice has also been reported by others (Thirunavukkarasu and Sakthisekaran, 2001; Pradeep et al., 2007). However, the levels of MDA were further significantly increased in post-NDEA stress treated group, which could be due to either increased production or decreased destruction of ROS, as the exposure to stress already causes a compromised status of the antioxidant system and thus decrease in anti-oxygenic potential in vivo. Reduced GSH is the major cytosolic thiol compound which plays important cellular functions including the destruction of hydrogen peroxide, lipid peroxides, and free radicals. The administration of NDEA caused a significant decrease in the levels of GSH, which may be responsible for the increased lipid peroxidation. NDEA administration after stress exposure caused a further decrease in the levels of GSH. This enhanced decrease of GSH in post-stress NDEA treatment suggests that the cells which are already deficient in thiol (-SH) groups undergo fast lipid per oxidation, as GSH is one of the guarding factors against oxidative stress. Enhanced lipid peroxidation associated with depletion of antioxidants is a characteristic finding in a variety of malignancies (Anis et al., 2001). The primary antioxidant enzymes SOD and CAT are involved in the inactivation of environmental carcinogens and direct elimination of toxic free radicals and electrophiles in vivo, which helps in the amelioration of the oxidative damage. The activities of both these free radical scavenging enzymes were found to be decreased in rats treated with NDEA (Yadav and Bhatnagar, 2007). The results of our study also indicate that the NDEA administration increases the susceptibility of hepatocytes to carcinogenesis by reducing the activities of SOD and CAT. The oxidative stress through the generation of ROS causes denaturation of protein/enzyme by the formation of protein carbonyls which might have caused inhibition of enzymatic activities (Höhn and Grune, 2014). A further significant decrease in the activities of SOD and CAT by CUS in NDEA-treated mice could be due to over utilization/less production of these enzymatic antioxidants to scavenge the products of lipid peroxidation and oxidative stress.

A large number of studies have established a correlation between cancer incidence and various disorders of GSH-related enzyme functions, alterations of GSTs being most frequently reported. A significant decrease in the activity of GSH dependent enzymes, GST and GR was observed in NDEA treated mice. This may be due to the decreased expression of these antioxidants during hepatocellular damage as previously described (Kweon et al., 2003). The further decreased levels of GST and GR as observed in the post-stress NDEA treated mice might have facilitated the NDEA induced liver carcinogenesis by decreasing its clearance from the system. The already compromised state of antioxidant level due to stress exposure might have further contributed in aggravation of the oxidative stress and NDEA induced toxicity. The diagnostic biomarker enzymes of hepatic damage GOT, GPT, and ALP are released into the circulation after cellular damage (Naik and Panda, 2007) and a rise of these marker enzymes in the serum causes increased permeability and necrosis of the cells (Al-Athar, 2004). In this study, we demonstrated that the administration of NDEA to mice leads to a marked elevation in the levels of GOT, GPT, and ALP which is indicative of hepatocellular damage, as also previously reported (Sadik et al., 2008). Further elevations of these enzymes in post-stress NDEA treatment could potentially be attributed to the release of these enzymes from the cytoplasm into the blood (Ritter et al., 2004) circulation after rupture of the plasma membrane due to increased free radical induced cellular damage. Both uric acid and glucose are also considered to be the major antioxidants found in blood plasma. The uric acid can act as an antioxidant either by binding to the radicals or by directly scavenging oxidizing species. It, therefore, inhibits lipid peroxidation (Muqbil et al., 2006). Glucose also acts as a scavenger of hydroxyl radicals (Halliwel and Gutteridge, 1990). The levels of these antioxidants were found to be decreased with increase in lipid peroxidation in plasma of mice exposed either to CUS alone or NDEA alone. The levels were further decreased when NDEA was given to the mice after exposure to stress, which shows that stress itself alters the antioxidant status, thus facilitating (Muqbil et al., 2006) and enhancing the NDEA induced liver carcinogenesis. Earlier studies from our laboratory have shown that CUS enhanced both the nephrotoxic and hepatotoxic potential of 7,12-dimethylbenz(a) anthracene in Swiss albino mice (Suhail et al., 2011), by altering the oxidative stress and compromising the antioxidant system, thus stress may act as a promoter of carcinogenesis by enhancing the pro-oxidant potential of carcinogens. Moreover, CUS also aggravated the development of skin tumors (in terms of their incidence, tumor yield, and tumor burden) in mice which further strongly supported our studies on the effect of stress on cancer promotion through DNA damage and modulation of oxidant/antioxidant system in vivo (Suhail et al., 2015). Thus, irrespective of the carcinogen used or the mode of application topical (Suhail et al., 2011, 2015) or oral as in the present study, CUS increased the toxic potential of the carcinogens.

All the above findings of oxidative stress were also supported by studies which showed CUS and NDEA caused significant damage to the DNA of lymphocytes and the liver cells as compared to controls. Post-CUS NDEA treatment showed further higher DNA damage in comparison with control, CUS alone or NDEA alone groups. The explanation for this may be that the chronic stress caused DNA damage within the cell either by altering the ability of the cells to repair DNA due to compromised antioxidant defense system (Bondi et al., 2008), or by causing oxidative stress and inhibiting apoptosis as observed by others also (Glaser et al., 1985; Kiecolt-Glaser et al., 1985; Adachi et al., 1993). The mechanisms that play roles in NDEA carcinogenicity in hepatocytes include DNA adduct formation followed by a gene mutation (Dyroff et al., 1986; Deal et al., 1989; Travis et al., 1991). Administration of NDEA generates lipid peroxidation products in general (Hietanen et al., 1987) and enhances the formation of the activated oxygen species in the preneoplastic nodules (Scholz et al., 1990) in the liver. The increased formation of reactive oxygen substances both in the circulation and in the liver tissues by NDEA administration either alone or after stress exposure further confirms an enhanced DNA damage by the chronic stress. Thus, the chronic stress plays an important role in the initial stages of liver carcinogenesis by compromising the antioxidant status, inducing oxidative stress and enhancing the DNA damaging potential of a carcinogen. Human body remains in a compromised antioxidant and enhanced oxidative status due to physical, physiological or psychological stress, and under this condition, any insult due to environmental carcinogens can aggravate the situation and may pave the way for carcinogenesis by enhancing the carcinogenic potential of any potent carcinogen irrespective of the mode of exposure.

Our results suggest a strong correlation between the observed alterations in the biochemical parameters, notably decreased antioxidant status and increased oxidative stress, due to the chronic stress exposure and the toxicity of environmental chemicals. Exposure to NDEA caused induction of oxidative stress and DNA damage by the generation and accumulation of large amounts of free radicals. Chronic stress further exacerbated this condition, thus putting at an increased risk of developing cancer. This study may offer a sensitive and useful approach for assessment of the effects of psychological stress on the carcinogenic risks of environmental chemicals (Bondi et al., 2008). Further studies may be aimed to combine the parameters of psychological stress with those of redox state, DNA-repair activity, and antioxidant defense system, so as to identify the relationship between psychological/physical stress and carcinogenesis at the molecular level to give a clearer picture.

This study was carried out after the protocol was approved by the Aligarh Muslim University ethics committee in accordance with the recommendations of the Indian national committee for the ‘Control and Prevention of Cruelty towards Experimental Animals’.

NB conceived and designed the study. NB, NS, GA, HK, and SH performed the experiments and wrote the manuscript. NB, AA, MA, and SF provided critical revision of the manuscript for intellectual content.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.