Investigation of the potential of high sugar and high caffeine to induce dna damage and oxidative stress in Drosophila melanogaster

In recent years, a significant increase in the global and especially national prevalence of diabetes has been observed (WHO 2021). This rise poses life-threatening risks to individuals while also imposing a considerable economic burden on countries. Diabetes leads to severe health complications such as cardiovascular diseases, kidney failure, blindness, and limb loss, thereby reducing quality of life and increasing mortality risk (Global Burden of Disease 2021; Diabetes Collaborators 2023). Continuous medication use, glucose monitoring, and management of complications impose both psychological and economic burdens on individuals (Seuring et al. 2015). Depression and anxiety are frequently observed comorbid conditions in individuals with diabetes (de Groot 2023). At the societal level, diabetes imposes a substantial economic burden on healthcare systems; approximately 10% of global healthcare expenditures are allocated to diabetes and its complications (International Diabetes Federation 2024). Furthermore, loss of workforce, early retirement, and decreased productivity amplify the economic damages (Kurkela et al. 2021). In low- and middle-income countries, diabetes exacerbates inequities in access to healthcare, thereby perpetuating the cycle of poverty (Parker et al., 2024). It is known that oxidative stress resulting from high sugar intake exerts neurotoxic effects on human health (Krawczyk 2023). This study aims to contribute novel insights to the literature regarding the potential toxicity of widely consumed caffeine in individuals with type 2 diabetes, thereby advancing the current state of knowledge on this topic. Within the scope of this research, "normal sugar" refers to the sugar dose present in the Lewis medium (Normal sugar; 11.7/128 ml), and the multiples of sugar correspond to multiples of this amount. Caffeine doses are expressed in mg/ml and denoted as K (K: Caffeine). Accordingly, the study investigated the neurotoxic effects of combined sugar and caffeine intake, as well as caffeine alone, in vivo on the brain cells of Drosophila melanogaster using alkaline single-cell gel electrophoresis (COMET assay). To assess feeding preferences, a two-choice feeding orientation analysis was conducted on adult D. melanogaster following larval exposure. Cellular oxidative stress levels were evaluated by measuring total antioxidant and oxidant status (TAS/TOS). The cytotoxic effects on adult brains were assessed via acridine orange staining based on membrane integrity. Additionally, lipid peroxidation (LPO) was examined to determine lipid degradation caused by oxidative stress induced by free radicals. According to the COMET assay results conducted on Drosophila brain cells, significant increases in DNA damage were observed at NŞ 0,5K - 2XŞ 0,1K, and 2XŞ 0,5K concentrations. The feeding orientation analysis revealed that, although individuals were raised on high-sugar diets from larval to adult stages, they continued to prefer high- sugar diets in the experimental setup while avoiding high caffeine doses. TAS/TOS analysis showed a decrease in antioxidant levels at NŞ 0,1K and NŞ 0,5K concentrations compared to the control group, while no significant change was observed in oxidant levels. Acridine orange testing indicated a statistically significant increase in cell death under high-sugar treatments compared to the control group. Although no significant differences in lipid peroxidation were detected between concentrations, the NŞ 0,5K concentration showed a slight difference compared to the control. Using a wide dose range of high sugar and high caffeine combinations (NŞ 0.1K, NŞ 0.5K, NŞ 1K, NŞ 2K, 2XŞ 0.1K, 2XŞ 0.5K, 2XŞ 1K, 2XŞ 2K, 4XŞ 0.1K, 4XŞ 0.5K, 4XŞ 1K, 4XŞ 2K, 8XŞ 0.1K, 8XŞ 0.5K, 8XŞ 1K, 8XŞ 2K), seven behavioral parameters were evaluated in Drosophila melanogaster, including larval weight, larval crawling, pupation site selection, adult weight, negative geotaxis, lifespan, and two-choice feeding preference, except for the two-choice feeding assay, all other behavioral experiments were performed in the 1st, 3rd, and 5th generations, and the results were compared to the control group, for the two-choice feeding assay, four doses (NŞ 0.1K, NŞ 0.5K, 2XŞ 0.1K, 2XŞ 0.5K) were tested and the results were obtained accordingly, in the larval crawling assay, a dose-dependent decrease in crawling distance was observed as generations progressed, in the larval weight assay, a generation-related weight loss associated with dose was observed, in the pupation site selection assay, as dose exposure continued over generations, the number of pupae decreased, and larvae pupated more frequently in the lower zones (D, E, BY), in the negative geotaxis assay, a generational and dose-related decrease in the number of climbing/flying individuals was identified, in the adult weight assay, weights were measured separately for female and male individuals, results showed a dose-related decrease in weight in both sexes in all generations except the 5th, in the 5th generation, although weights remained lower compared to the control, an increase was observed compared to earlier generations, in the lifespan assay, while increased lifespan was observed in sugar-associated 0.1K groups over generations, other dose groups exhibited a reduction in survival, in the two-choice feeding assay, although the individuals were exposed to triple sugar concentrations, their preference remained for sugar-rich diets, and they avoided caffeine-containing foods, when exposed to two different caffeine doses, individuals preferred the lower caffeine dose.