Gallic acid rescues uranyl acetate induced-hepatic dysfunction in rats by its antioxidant and cytoprotective potentials

The significant increase in plasma AST and LDH activities mirrors observations from a previous study [4]. In our experimental model, the oxidative burden triggered by UA led to hepatocyte cytolysis, causing the release of cytosolic enzymes into the bloodstream. The elevation in AST activity can be linked to increased production of Krebs cycle intermediates, contributing to fueling gluconeogenesis to meet cellular metabolic demands while maintaining antioxidative capacities to counteract redox imbalances [26]. The increased LDH activity is associated with lactic acidosis, which is implicated in apoptosis through opening the mitochondrial permeability transition pore and inducing cytosolic Ca²⁺ bursts that activate caspases [27]. Conversely, GA effectively normalized plasma AST and LDH activities, consistent with findings in paraquat-induced hepatotoxic rats [28]. The hepatoprotective mechanisms of GA involve hindering the access of oxygen-derived species to the lipid bilayer, reversing oxidative/nitrosative-mediated membrane disruptions, and stabilizing tight junctions and epithelial barriers [29‐31].

The UA-associated hyperglobulinemia in our experimental irradiated rats aligns with increased serum immunoglobulin levels observed in orally supplemented mice [32]. This suggests enhanced B cell differentiation to boost specific immunity against xenobiotic contamination or potential impairment in the ability of the liver to clear immunoglobulins from circulation [33, 34]. As part of a compensatory response to reactive damaging molecules, accelerated protein generation supports the biosynthesis of antioxidants and cytoprotective agents [35]. The hyperproteinemia observed in the UA group contradicts findings by Zimmerman and colleagues [36], who reported no significant change in total protein. However, it could serve as a symptomatic marker of hepatic dysfunction [37]. Hyperproteinemia may instigate generation of reactive free radicals and activation of programmed cell death through the endoplasmic reticulum-calcium ion signaling pathway [38] following UA exposure. It was hypothesized that the increase in albumin and ALT by hepatocellular injury was masked by its reduced production due to extensive fibrosis [26, 39], resulting in an insignificant change in ALT and albumin following UA exposure.

Similar to gamma-irradiated rats [40], our experimental irradiated model displayed a marked increase in plasma glucose levels, attributed to mobilization of hepatic glycogen reserves as confirmed histologically. Reduced renal glucose excretion, decreased beta cell number, and impaired glucose uptake [41‐43] contribute to this hyperglycemic state. Hyperglycemia-induced overproduction of reactive oxidants may disrupt endothelial tight junctions and the barrier function, leading to leakage of blood from vasculature into surrounding tissues [44]. UA-associated hyperglycemia appears to down-regulate gene expression of Nrf2 and its regulators [45]. Additionally, it down-regulates anti-apoptotic proteins, up-regulates pro-apoptotic factors, and promotes cytochrome c translocation from mitochondria to the cytosol [46]. In contrast, GA supplementation restored glucose homeostasis as demonstrated by Variya and colleagues [47], achieved by delaying intestinal glucose absorption, enhancing beta-cell insulin secretion, and encouraging glucose uptake and peripheral insulin sensitivity [48].

The disturbances in lipid profile observed in the UA group are similar to the findings in gamma-irradiated rats [49]. Elevated activity of hepatic metabolizing enzymes responsible for fatty acid synthesis and mobilization contributes to radiation-induced hyperlipidemia [49]. Up-regulation in the transcript levels of sterol regulatory element-binding protein 1c might participate in this effect [50]. TG enrichment of HDL particles, enhancement of hepatic lipase activity, and inhibition of hepatic production of apolipoprotein A-1 may be responsible for the drop in plasma HDL-C [51]. Damage to the pancreas is a leading cause of inhibiting lipoprotein lipase [52], closely associated with the observed lipoprotein patterns [53]. A two-way relationship exists between hyperlipidemia and hyperglycemia. Hyperlipidemia promotes insulin resistance by blocking insulin signals and destroying pancreatic beta cells, giving rise to hyperglycemia [54]. As a consequence of excess glucose loading, lipid metabolism is impaired. For instance, glucose can be converted to fatty acids and cholesterol through de novo lipid biosynthesis pathways, and excessive lipids are secreted in lipoproteins or stored in lipid droplets [55]. Hyperglycemia can predispose to hypercholesterolemia by up-regulating 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and hamper fecal cholesterol excretion and bile acid biomanufacturing [56, 57]. Peroxidation of membrane phospholipids exacerbates cholesterol biogenesis in the liver and other organs through overgeneration of peroxides and disruption of membrane structure-function attributes [58]. Hyperlipidemia induces hepatic oxidative stress, inflammation, and apoptosis [59]. The current GA intervention dosage and duration may be insufficient to counter UA-associated hyperlipidemia, as observed in atherosclerosis-prone apolipoprotein E knockout mice fed a high-fat Western-type diet [60]. Factors affecting gut microbial community and xenobiotic detoxification systems may play a dominant role in modulating the stability, absorption, and metabolism of phytochemicals [61].

The substantial increase in lipid peroxidation end products after UA intoxication concurs with findings by Yuan et al. [4]. UA stimulates excessive free radical production while inhibiting the intracellular redox stabilizing network in rat hepatocytes [62]. This impairment not only affects polyunsaturated fatty acids but can also impact other biological macromolecules, thereby affecting cellular membrane levels and subcellular components [63]. Our work, along with others [64], confirms the ability of GA to counteract the lipid peroxidation cascade in hepatic tissues due to its free radical scavenging properties. GA suppresses the Fenton reaction, which reduces the production of free radicals and the amount of iron available to combine with oxygen to initiate lipid peroxidation [65].

Matched with the depletion of NO in the testicular tissues of UA-exposed rats [8], our finding revealed a remarkable exhaustion of hepatic NO owing to a reduction in NO-secreting cells and inducible NO synthase activators and elevation in NO inhibitors [32]. Reduced NO bioavailability could result from its binding with superoxide radicals to form peroxynitrite or uncoupling of nitric oxide synthase under oxidative stress, further exacerbating the redox imbalance [66]. Disturbances in lipid metabolism in our irradiated model could contribute to NO depletion through mechanisms including L-arginine exhaustion (a key player in NO synthesis), NO synthase dysfunction, increased NO turnover, limited vascular response to its vasodilatory effects, and impaired translocation to target tissues [67]. The elevation in apoptotic signaling and reduction in cell proliferation capacity often correlate with a deficit in NO formation. This is evident from the cytoprotective properties of NO through S-nitrosylation of apoptotic mediators [68]. In contrast, GA supplementation increased hepatic NO levels, surpassing even control levels. This effect is due to the ability of GA to slow NO turnover and enhance endothelial NO synthase phosphorylation [69, 70]. Increased NO levels activate the pentose-phosphate pathway [71], a major NADPH producer that regenerates reduced GSH from its oxidized form. The elevation in NO levels correlates with the increase in SOD activity, suggesting a causal link. As SOD catalyzes the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide, heightened SOD activity clears superoxide anions, thus preserving NO bioavailability [72]. Additionally, NO is essential for up-regulating SOD expression, preventing superoxide radical-mediated NO degradation [73].

Similar to the findings of Hao et al. [74] and Pourahmad et al. [62], GSH redox network was altered in the UA group. This outcome indicates a failure in a critical component of the xenobiotic detoxification system [75], rendering the hepatic microenvironment more susceptible to the radiological hazards of UA. Lactic acidosis prompts metabolic reprogramming to enhance NADPH synthesis, shifting the glutathione redox couple towards the oxidized form to counter reactive oxidative stress [76]. Moreover, utilization of glutamine for ATP production under acidic stress contributes to the depletion of other glutamine-related metabolites, including GSH [76]. Reactive oxidant generation caused by UA triggers GSH oxidation and inactivation of GSH-related enzymes [77]. GST eliminates lipid peroxidation end-products and contaminants-derived electrophilic compounds [78, 79], thereby preventing cell membrane damage. The reduction in GST could be due to the down-regulation of its gene expression [80]. GSH is necessary for ensuring the continuation of thiol group reduction in mitochondrial membrane proteins [81]. When these thiol groups are oxidized, the pore complex undergoes structural modifications, resulting in a mitochondrial permeability transition that is a leading factor in both necrosis and apoptosis mechanisms [82]. Restoration of hepatic GSH redox cycle in the GA + UA group is compatible with what happened in doxorubicin-induced hepatotoxic [77] and streptozotocin-induced diabetic rats [65]. The increase in hepatic GSH levels in UA-irradiated rats pre-supplemented with GA is attributed to the up-regulation of gamma-glutamylcysteine synthetase, a rate-limiting enzyme in GSH biosynthesis [83]. Activation of Nrf2 results in increased transcript abundance of downstream antioxidants-related genes, including those belonging to the GSH redox system [84].

Total antioxidant capacity (TAC) provides a holistic view, accounting not only for the sum of individual antioxidants but also for their complex interactions [85]. The normalization of TAC reflects the ability of GA to restore the overall body’s redox balance. The improved redox potency of hepatic tissue in the GA + UA group is attributed to increased transcript levels of antioxidants and scavenging of free radicals [86, 87]. This is supported by the increase in Nrf2 immuno-expression, a crucial transcription factor that plays a pivotal role in defending against peroxidative damage by up-regulating various enzymatic antioxidants. The Nrf2 signaling pathway is a critical mediator in controlling the transcription of numerous antioxidant genes, including enzymes involved in GSH and SOD synthesis [88]. GA disrupts the interaction between kelch-like ECH-associated protein 1 and Nrf2 in drug-induced hepatic dysfunction, leading to increased nuclear translocation of Nrf2 [83]. Sirtuin 1 overexpression resulting from GA supplementation facilitates Nrf2 nuclear translocation, stabilizes Nrf2 protein expression, and enhances nuclear accumulation, DNA binding activity and transcriptional function of Nrf2 [89, 90]. Targeting Nrf2 may offer a promising therapeutic strategy to enhance cellular stability against redox imbalances, a key factor in driving and exacerbating radiation-associated hepatic damage.

The hepatic histoarchitectural deteriorations induced by UA exposure are consistent with other reports [4, 91]. These cellular changes could arise from mitochondrial dysfunction and disruption of oxidative phosphorylation [1]. Vacuolated cytoplasm in the liver could result from dysregulated fatty acid metabolism, leading to neutral fat accumulation, which gets dissolved during tissue preparation, leaving empty unstained vacuoles [92]. Karyolysis in hepatocytes, similar to uranium-contaminated mice [93], is attributed to endonuclease activity secreted by Kupffer cells, causing destructive fragmentation of genomic material [94]. The heightened release of reactive oxygen species, as indicated by increased MDA levels in the UA group, drives the excessive formation of extracellular matrix proteins ensuring optimum conditions for hepatic fibrosis [95]. Hyperlactemia resulting from increased LDH activity triggers transforming growth factor-beta, leading to fibroblast differentiation [96]. The marked occurrence of apoptotic hepatocytes following UA contamination aligns with findings in testicular germ cells [8]. UA triggers genotoxic damage indirectly through single-strand breaks, facilitated by oxidative DNA damage via Fenton redox reactions, and directly through covalent binding to DNA [97]. GA excretes antifibrotic activity by reducing hepatic pro-fibrogenic cytokines and blocking hepatic stellate cells activation and proliferation [98]. The anti-apoptotic effect of GA against UA-induced hepatotoxicity corresponds to its protection against ultraviolet radiation-induced damage in zebrafish and human keratinocytes [99]. Scavenging free radicals, reducing transcript levels of Bax and caspase-3, increasing Bcl-2 transcript levels, and enhancing genomic repair [9, 100] underlie the cytoprotective properties of GA.