Diabetes mellitus is a chronic metabolic disorder disease, widely spread in human, characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia was found to increase the production of oxygen free radicals (OFRs) that is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels [1, 2]. Under normal conditions adequate protection is provided by the antioxidant systems, a number of major cellular antioxidant defense mechanisms exist to neutralize the damaging effects of these free radicals. If the OFRs are not quenched by antioxidants, these highly reactive compounds will react and potentially alter the structure and function of several cellular or extracellular components, such as cell membranes, lipoproteins, proteins, carbohydrates, RNA and DNA [3-6]. The increase in the level of OFRs in diabetes was reported to be due to their increased production and/or decreased destruction by non-enzymic and enzymic antioxidants. Catalase (CAT), superoxide dismutases (SODs), and glutathione (GSH) is the major enzymic and non-enzymic anti-oxidants that remove and protect the cells from the free radicals [7, 8].

Currently available synthetic antidiabetic agents produce serious side effects, such as hypoglycaemic coma and hepatorenal disturbances [9]. Moreover, they are not safe to be used during pregnancy [10]. Recently, it has been assumed that many dietary plants contain numerous types of antioxidants with different properties. Many of these antioxidants cooperate in oxidative stress reduction. To test this hypothesis, it was useful to identify dietary plants with high total antioxidant content.

Several nuts are among the dietary plants with the highest content of total antioxidants. Vinson and Cai [11] reported that a gram of walnuts (Juglans regia L.) contains nearly 70 units of polyphenols, a form of antioxidant more than any of the other nuts. The polyphenols were also up to 15 times more potent than those found in other sources like vitamin E. Walnuts extracts are superior in binding with lower density lipoprotein (LDL) and inhibiting its oxidation than other nuts. The 14 walnut polyphenols have superoxide dismutase (SOD)-like activity with EC50 21.4-190 μM and a remarkable radical scavenging effect against 1, 1-diphenyl-2-picrylhydrazyl [12].

Pharmacogenomics can be defined as the sum of the word’s parts: the study and application of genetic factors related to the body’s response to drugs, or pharmacology. Scientists are increasingly learning more about the interaction between drugs and human genetics in order to develop therapeutic medicine more efficient without adverse effects. At the molecular level, the metabolite can bind to the protein’s active site, which can include ligand-binding sites, conformation-altering sites, or catalytic sites. This effect can then be propagated through biochemical pathways to produce a cellular and finally, systemic physiological effect [13, 14].

Although various phytochemical constituents and diverse medicinal activities have been attributed to walnuts, according to our knowledge, the biochemical and pharmacogenomics studies have not been carried out to study whether walnuts can protect against oxidative stress induced diabetes in different tissues. This study aimed to evaluate the adverse effect of walnuts mixture diet on streptozotocin-induced diabetic mice. Comprehensive systems of biochemical markers and genomic screening were used to achieve this task. Assessment of antioxidant biomarkers activity [Superoxide dismutases (SOD), Catalase (CAT), Glutathione (GSH) and lipid peroxidation], expression level of SOD, CAT and GPx genes and DNA damage using DNA fragmentation, micronucleus and comet assays were investigated.

Achievement of effective and safe therapeutic drugs is the main target of drug discovery. Recently, scientists work on building a therapeutic regimen that optimizes the efficacy of the therapy while at the same time limiting potential adverse or toxic side effects. Natural substances are the main sources of therapeutic drugs that provide less adverse effect than synthetic therapeutic drugs [9]. Walnuts contents have several antioxidants, which can fight the free-radical damage that contributes to cancer, heart disease, premature aging and cell death [25-27].

The Study of pharmacogenomics and biochemical influences of walnuts on diabetic tissue complies with drug discovery focus on effective treatment without/less adverse effect. The effect of walnuts antioxidant on induced diabetic mice was investigated. Type 1 diabetes was induced by i.p. injection of STZ for 3 successive days. Mice were considered diabetic when the blood glucose concentrations increased to 200 or more mg/dl. Experimental animals were divided into four groups (negative and positive control, diabetic group and diabetic group fed on walnuts diet). Walnuts influence on control and diabetic tissues was assessed using biochemical markers, genes expression and DNA damage assays.

Biomarker is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Superoxide dismutase (SOD), Catalase (CAT) and Glutathione (GSH) reported to be used as biomarker indices to evaluate the formation of free radicals. SOD constitutes the first line of defense against reactive O2 species (ROS). Specialization of function among the SOD may be due to a combination of influence of subcellular location of the enzyme and upstream sequences in genomic sequence [28]. CAT is major function within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the electron transport pathway [29]. GSH is often referred to as the body's master antioxidant and essential component to the body's natural defense system [30]. In patients with diabetes mellitus, free radical-induced damage arises from an imbalance between pro-oxidant and antioxidant systems [31]. The results of diabetic mice fed on basal diet showed significant decrease of SOD, CAT and GSH levels in liver and kidney tissues, while diabetic animals fed on walnuts diet showed marker elevations in SOD, CAT and GSH to the normal levels of control groups (Table 2). MDA level increased in liver and kidney tissues of diabetic group (6.26±0.08 and 2.57±0.22, respectively), while walnuts treated diabetic animals afforded a significant reduction in MDA level (3.13±0.08 and 1.55±0.08, respectively). MDA level increase in diabetes traced back to the hyperglycemia which induces carbonyl stress and lead to increased lipid peroxidation and oxidative damage due to increase of peroxy radicals and hydroxyl radicals [32-34]. Our results of biochemical analysis suggested that the walnuts antioxidant substances act to quench the oxidative stress in diabetic tissues, which could not be removed by diabetic mice's antioxidant systems.

Applications of pharmacogenomics are of interest to industry, clinicians, academics, and patients alike. For the biopharmaceutical industry, pharmacogenomics can improve the drug development process through faster and safer drug trials and the early identification of drug responders, non-responders, and those prone to adverse events [14]. Gene expression is combined with metabolic schemas to understand how pathways are changed under varying conditions. The expression levels of SOD, CAT and GPx genes in diabetic mice of our study were in agreement with recent studies reporting that the mRNA expressions of SOD and CAT decreased, while the GPx increased with higher plasma glucose concentrations in diabetes [7, 35, 36]. The results of expression levels of oxidative stress genes studied in kidney tissues of mice fed on walnuts diet showed that the SOD and CAT genes expression levels were compensatory increased to normal level (Table 3), while GPx gene over-expression in diabetic mice (2.5± 0.31) decreased with walnuts treatment (2.1± 0.26). No significant differences were found between diabetic mice fed on walnuts diet and control groups. These results suggested that the expression levels of these genes were associated with the respective activities of antioxidant enzymes tested and walnuts treatment act to improve genes expression to normal.

Regulatory authorities all over the world require data on the genotoxic potential of new drugs chemical compounds, as part of the safety evaluation process. Genotoxicity assays became an integral component of regulatory requirements. It can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage by various mechanisms. Comet assay is a sensitive technique for measuring and analyzing DNA breaks in various mammalian cells [20, 37, 38]. It is widely used in areas of research ranging from biomonitoring and studies of DNA damage processes to routine assessments of genotoxicity [39-41]. DNA damage with the frequencies of comet untailed, tails, tail moment, %DNA in tail and tail length have been detected in all tested animals. The results illustrated that the walnuts reduce the DNA damage in group IV compared to group III. The comet assay results suggested that the walnuts treatment decrease cells necrosis.

DNA fragmentation, used to study apoptosis in cell populations. DNA damage in mice liver was detected by visualization of the DNA of tested animals upon gel electrophoresis (Fig. 4). The gel represented the genomic DNA degraded in different groups. The smear of DNA down in lane3 (diabetic mice) indicated that the DNA genome of diabetic mice degraded into thousands of smaller fragments. Darker bands present in lanes 1, 2 and 4 (Fig. 4) indicated that these samples contained less apoptosis and more live cells expressed in clear dark DNA band. The DNA fragmentation results clarified that the walnuts treatment in diabetic mice decreased the DNA fragmentation.

Micronucleus test is widely employed in different areas as biological monitoring. Micronuclei are small chromatin containing bodies arising from chromosome fragments or whole chromosome that were not incorporated into daughter nuclei following mitosis [42]. The result of MN frequency showed higher significant difference between Streptozotocin treated animals and control groups (Table 6). The diabetic animals in group IV fed on walnuts diet showed significant decrease in MN frequency compared to diabetic animals fed on basal diet. The micronucleus assay results confirmed the positive influence induced by walnuts treatment in diabetic animals.

Evaluating the results of negative and positive controls clarified that there were no significant differences between the two groups at the metabolic activity and expression levels of tested genes. While using DNA fragmentation, micronucleus and comet assays showed significant differences at P < 0.05 between negative and positive control. These results could be referred to the accuracy of these assays that precisely measure the minor adverse effect of compounds. This level of adverse effect on DNA damage could be withdrawn with normal DNA repair processes [43].

In conclusion, pharmacogenomics and biochemical assays have potential value for assessing and monitoring the effectiveness and safety of pharmaceutical and medicinal therapy. The results suggested that walnuts could act as diabetic therapy without adverse effects at metabolic activity and molecular levels in diabetic mice.

The author(s) confirm that this article content has no conflicts of interest.