The present work focuses on testing another mechanism for the induction of hepatic aging by D-galactose or γ-irradiation following the study by Habieb et al. (Habieb et al. 2021). Biomarkers of oxidative stress were used with those essential elements in the biological system
The results of this study clearly showed that a rise in the WBC counts exists, followed by a substantial decrease in the G6PD activity. Glycation end-products may result in the elevation of white blood cell counts by D-galactose (Hofmann et al. 1999; Yang et al. 2007). Moreover, D-galactose interacts with the amino protein group creating derivatives called advanced glycation end-products. These glycation end derivatives have been shown to improve some angiogenic and inflammatory cytokine expression, including vascular endothelial growth factor, TNF-α, IL-6. Moreover, this method could be the reason for leukocyte count activation and elevation (Costa et al. 2007).
Current research has shown an increase in platelets based on βOHB administration, which reflects an improvement in the process of differentiation by βOHB per se. Platelet count elevation could be associated with increased liver production of thrombopoietin, which in turn may be responsible for the proliferation of megakaryocytes and their conversion to platelets (Lee and Bergmeier 2017; Subenthiran et al. 2013).
Exposure to γ-irradiation significantly increased MCV. This result is consistent with Abdelhalim et al. (Abdelhalim et al. 2015) who indicated that a change in morphology and deformation of RBCs can be attributed to an increase in the size of RBCs by γ-irradiation, which has been confirmed by a slight increase in red distribution width.
A significant decline in RBCs, WBCs, platelets, and hemoglobin content in irradiated rats could be due to erythrocyte precursor destruction in bone marrow (Ashry and Hussein 2007). In addition, decreased production of kidney erythropoietin hormone, which is essential for the production of RBCs or erythropoiesis, can be attributed to increased destruction of RBCs by γ-irradiation (Hassan et al. 2016). Free radicals are known to be responsible for RBCs because they contain extraordinary membrane content of polyunsaturated fatty acids (PUFAs) and oxygen transport linked to redox-active hemoglobin molecules, which are efficient promoters of activated oxygen species (Ebrahimzadeh et al. 2009). Exposure to γ-irradiation significantly increased MCV. This result coincides with Abdelhalim et al. (Abdelhalim et al. 2015) who suggested that a shift in morphology and deformation of RBCs could be due to an increase in RBC size by γ-irradiation.
The cellular components of the blood are largely susceptible to oxidative stress because the phospholipid bilayer membranes have a high concentration of PUFAs. The decrease in the number of WBCs reported in irradiated rats may therefore be the product of lipid peroxidation caused by radiation and its detrimental impact on the phospholipids of their cell membranes. The drop in hemoglobin content may be related to a decrease in red blood cell counts (Chew and Park 2004; Malhotra and Srivastava 1978).
Glucose-6-phosphate dehydrogenase (EC 126.96.36.199), which catalysis glucose-6-phosphate oxidation to 6-phosphogluconolactone and finally to ribose-5-phosphate, is a major rate-limiting enzyme of the pentose phosphate pathway (PPP). This results in NADPH-equivalent reduction biosynthesis to satisfy cellular needs for biosynthesis of fatty acid reduction and maintenance of cellular redox balance (Maurya et al. 2016).
γ-irradiation has a more pronounced effect on G6PD decline activity compared with the decrease induced by D-galactose. The PPP inhibition can then render the exposed RBC more vulnerable to oxidative damage (Demirdag et al. 2015). These changes can result from the vulnerability to oxidation induced by D-galactose or γ-irradiation of free sulfhydryl groups (-SH) necessary for its activity (Giblin et al. 1979). Moreover, D-galactose or γ-irradiation alternation of the redox state of the RBCs subsequently decreases NADPH formation, which in turn inactivates the G6PD activity (Pari and Venkateswaran 2003). Increased RBC exposure to ionizing radiation resulted in a substantial decrease in G6PD activity by γ-irradiation, thereby decreasing survival time (Agarwal et al. 2007). These findings were consistent with those of Van Heyningen et al. (Van Heyningen et al. 1954) who revealed that ionizing radiation such as X-irradiation affected -SH-based enzymes than those enzymes that do not have free -SH groups for operation. Thus, these findings suggest a general improvement in hematological parameters by βOHB by modulation of G6PD activity and decreasing oxidative stress.
Oxygen-derived free radicals enhance membrane lipid peroxidation, enzyme inactivation, DNA fragmentation, and apoptosis activation resulting in disturbance in the living cells (Speakman and Selman 2011). MDA is the end product of lipid peroxidation, which is considered a significant biomarker. Additionally, antioxidant supplementation has an important role to reduce the aging process (Koyama et al. 2013). There are many enzymes in the body such as SOD and glutathione peroxidase which are responsible for scavenging superoxide anion and hydrogen peroxide to prevent ROS-induced damage. Thus, it has been important to determine the oxidative stress status including MDA, NO, GSH levels and SOD activity. Our data recorded that βOHB and D-galactose or γ-irradiation significantly reduced the oxidative stress in the aged rat by restoring GSH levels and SOD activity in the liver and decreasing MDA level and modulating the trace element alterations, supporting the βOHB mechanism of action and the aging theory of oxidative stress.
The supplementation with DL-β-hydroxybutyrate dramatically increased MDA levels in the liver. However, this finding was not predicted. Thus, this is likely to be because the daily ingestion of ketone salt could cause recurrent bouts of oxidative stress to the liver and this finding requires further mechanistic analysis (Lu et al. 2018). The unusually high level of MDA caused by βOHB alone correlates with Milder et al. (Milder et al. 2010), which could be attributable to the acute increase of hydrogen peroxide (H2O2) and 4-hydoxyneonal, an electrophilic lipid peroxidation end-product known to activate nuclear factor erythroid 2-related factor 2 (Nrf-2) detoxification pathway. Moreover. Nrf-2 can upregulate GSH biosynthesis and is a primary response to cellular stress. In addition, another possible explanation for this increase in MDA by βOHB per se may be due to lipid peroxidation mediated by macrophages of liver hepatocytes as an NADPH oxidase (NOX) enzyme upregulated by βOHB that acts as a mediator of the development of reactive oxygen species (Kanikarla-Marie and Jain 2015). The results coincide with Anilkumar et al. (Anilkumar et al. 2008) who reported that high ketone levels were reported to be associated with increased oxidative stress in cell models. Moreover, Baffy (Baffy 2009) correlated an increase in the burden of oxidative stress on macrophage recruitment along with liver resident Kupffer cells, which subsequently raises the levels of hepatic MDA. Furthermore, the findings of this study are in line with the results reported by De Almeida et al. (De Almeida et al. 2010), who showed an increase in protein oxidation and lipid peroxidation in animal model rat livers subjected to acetone-induced ketosis via a free radical dependent mechanism.
In the hippocampus of rats fed a ketogenic diet, Milder et al. (Milder et al. 2010) reported an improvement in H2O2 development and lipid peroxidation. In addition, chronic βOHB exposure has also been shown to increase the production of ROS in cardiomyocytes (Pelletier and Coderre 2007).
Liver SOD activity was retained in the βOHB group. These data indicate that βOHB initially generates mild oxidative stress, which through redox signaling can systematically activate the Nrf-2 pathway, leading to an adaptive redox response to oxidative stress (Lu et al. 2018). A significant decrease was not found in SOD activity along with D-galactose administration or γ-irradiation exposure, unlike other research conducted in this field. However, the cellular antioxidant SOD activity was significantly elevated by D-Gal or γ-irradiation exposure. This somewhat contradictory outcome is still not entirely clear, but this outcome can be explained in part by MDA stimulation at a sublethal concentration that induces adaptive response and increases cell tolerance primarily through SOD induction through transcriptional activation of the signaling pathway linked to nuclear factor erythroid 2–factor-2 (Nrf-2) signaling pathway, thereby attempting to protect hepatocyte against forthcoming oxidative stress (Pahl and Baeuerle 1994). The aforementioned findings can be elucidated by the existence of a better equilibrium between free radical scavenging enzymes and the development of ROS in young tissue, which is very necessary for oxidative stress cell resistance. The findings may highlight the significance of the animal's starting age in relation to D-Gal-mediated depletion of antioxidants due to oxidative stress (Xu et al. 2009). The previous findings contradicted those of Liu et al. (Liu et al. 2013), who suggested a significant decrease in SOD activity which is a characteristic of various ages at the start of the D-galactose treatment.
No substantial improvement in GSH or NO levels was noted it was predicted that D-Gal would decrease GSH levels and increase the concentration of NO. An unchanged GSH level was followed by D-Gal-treated rats, leading to a satisfactory explanation that either GSH's response to increased lipid peroxidation is restricted or a lack of improvement in the GSH level is a cause for elevated levels of oxidative stress. The absence of adequate changes in GSH levels under conditions of increased ROS can impair the antioxidant system's capability, leading to increased oxidative stress (Hadzi-Petrushev et al. 2015). In addition, oxidative stress stimulation at a sublethal concentration, which induces adaptive response and increases cell tolerance primarily via GSH induction through transcriptional Nrf-2 signaling pathway activation, protects the hepatocytes from potential oxidative stress (Pahl and Baeuerle 1994). Furthermore, Wei and Lee (Wei and Lee 2002) suggested that no major changes in GSH or NO associated with D-Gal in the community of young rats could be found in the light of compulsory attention to developmental changes and the impact of aging on the antioxidant enzyme. Moreover, the aforementioned findings can be explained by the existence of low development of ROS and a better balance between free radical scavenging enzymes in young tissue, which is very important for the oxidative stress resistance of cells. The findings may highlight the significance of the animal's starting age to D-Gal-mediated depletion of antioxidants due to oxidative stress (Xu et al. 2009). The previous results were in contradiction with the findings of Liu et al. (Liu et al. 2013) who indicated that a significant decrease in GSH levels exists, which is a characteristic of various ages at the start of D-Gal treatment.
Nonsignificant increases in NO levels were observed in the D-Gal-treated group in the present study, which could be interpreted as resulting from increased NO oxidation by ROS. Consequently, nitrotyrosine generation in combination with tyrosyl radical or peroxynitrite formation in combination with superoxide ion reduces the availability of NO levels (Ferrer-Sueta et al. 2018; Reiter et al. 2000).
Cu is considered as a cofactor of many enzymes, but free ionic copper is cytotoxic because it mediates the development of the extremely reactive hydroxyl radical through the Fenton reaction. Therefore, it is believed to cause premature senescence and oxidative stress.
After γ-irradiation, the reduction of hepatic copper may be related to the excess of its usage by cuproenzymes which reduce the oxygen to water or hydrogen peroxide (Kotb et al. 1990) or due to de novo syntheses of Cu-SODs and catalase that inhibit the development of superoxide ion, H2O2, and hydroxyl radicals (Fee and Valentine 1977). Nada et al. (Nada et al. 2012) suggested that the decrease in copper levels is linked to the radiolytic loss of cofactors of essential metalloelements that record the 20% loss in rats post irradiation of both Cu- and Zn-dependent SODs.
Aging is well known to induce Fe accumulation in many tissues in vivo, which is associated with the pathology of many age-related diseases (Xu et al. 2008; Zecca et al. 2001). These alterations may be as a result of iron homeostasis deregulation at the cellular level. But, the mechanism needs to be further investigated. This was confirmed by the previous result of Killilea et al. (Killilea et al. 2003) who stated that the total iron content in the fibroblasts was presented to rise exponentially during cellular senescence, getting tenfold greater levels of Fe compared to young cells. Moreover, low-dose exposure to H2O2 lead to early fibroblast cell senescence and enhanced iron accumulation related to senescence. This accumulation may also associate to the amplified amounts of oxidative stress and the decline of cellular function that describe senescent cells.
D-galactose induced a significant increase of iron in the liver. These findings are in complete agreement with Beregovskaia et al. (Beregovskaia et al. 1988) and Nada et al. (Nada et al. 2008) who revealed that Fe level elevation could be correlated with bone marrow's inability to use the Fe available in the diet and released from damaged red cells due to oxidative stress caused by D-Gal. The increased level of iron may be due to proteolytic modification of oxidative stress-induced ferritin (García-Fernández et al. 2005) and transferrin (Trinder et al. 2000). Fe overload is associated with liver damage, characterized by a large accumulation of iron in hepatic parenchymal cells, resulting in fibrosis and ultimately hepatic cirrhosis (Pietrangelo 2016). Increased iron levels by D-Gal agree with Xu et al. (Xu et al. 2012) who suggested that Fe accumulation is generally accepted as an essential characteristic of the aging process, particularly in postmitotic tissue. Consequently, both heme-Fe and heme biosynthesis decrease significantly with aging (Mancuso et al. 2013).
Zn is an important cofactor that maintains the activity of several proteins, such as metallothionein and Cu/Zn SOD that play a role in combating oxidative stress. Zn is also a cofactor for proteins involved in mediating the reaction and repair of DNA damage, such as the p53 tumor suppressor protein (Theocharis et al. 2004). Suboptimal Zn intake can therefore encourage oxidative stress generation and single- or double-strand DNA breaks comparable to DNA damage caused by radiation (Blount et al. 1997). Thus, precise regulation of cytosolic Zn buffering is important to preserve the redox state of the cell because both increased and decreased Zn levels induce oxidative stress (Maret 2006).
The results of the current study showed a significant decrease of Zn in the liver by D-Gal or γ-irradiation which could be due to molecular process disruption that allows optimum Zn levels to be preserved within the cells (Colvin et al. 2010). In addition, increased Zn efflux transporter ZnT-1 levels in the hepatic cell membrane may lead to excess Zn efflux from the liver, resulting in lower levels of zinc in the hepatic cell cytosol (Sekler et al. 2007). Moreover, lower Zn levels in the liver may be mediated by the incorporation of zinc in superoxide dismutase with antioxidant function because the balance between oxidative stress and performing antioxidative defense preserves proteostasis and cellular function and also affects the rates of aging and telomere shortening. Furthermore, the induction of superoxide dismutase plays a significant role in cell protection against an excessive amount of ROS and acted against oxidative stress, thus constituting the main system of protection against oxidative stress induced by D-Gal or γ-irradiation (Andrews 2000).
Oxidative damage caused by either D-Gal or γ-irradiation may be due to a decline in Zn levels because increasing evidence points to Zn as a positive regulator of autophagy. In vitro studies have consistently shown that both basal and induced autophagy is important for Zn (Liuzzi and Yoo 2013). This was confirmed by the fact that high doses of Zn were shown to induce autophagy in MCF-7 breast cancer cells in culture medium (20–200 µM; (Hwang et al. 2010), astrocytes (Park et al. 2011), and also in human hepatoma cells (Liuzzi and Yoo 2013). In addition, Zn depletion caused by treatment with TPEN or Chelex-100 cell-permeable Zn chelators has been able to suppress autophagy (Liuzzi et al. 2014). Moreover, a possible Zn function of Zn was suggested as a modulator of inflammation associated with the senescence-associated secretory phenotype (Malavolta et al. 2015).
Zn deficiency was observed during aging and increases the suspicion that a possible relationship between Zn and cellular senescence-related hepatic injury may exist. This was explained by the study of Rudolf and Rudolf (Rudolf and Rudolf 2015) who found that colon cancer cells cultivated in a low Zn environment for 6 weeks resulted in morphological changes and typical markers of senescence, while Zn supplementation induced increased ROS formation, causing senescence in vascular smooth muscle cells. Zn also declined the antioxidative ability of the cell by downregulating catalase expression (Patrushev et al. 2012). Additionally, Carrı̀ et al. (Carrı̀ et al. 2003) stated that an imbalanced intracellular Zn homeostasis mediates both oxidative damage and neuronal cell death in neurodegenerative diseases. So, oxidative damage and hepatic cellular senescence may be stimulated by unbuffered controlling mechanisms of intracellular/ cytosolic zinc.
Concerning Zn concentrations in liver tissue, irradiation caused a decrease in liver Zn levels. Similar findings were obtained by Ali et al. (Ali et al. 2012) who suggested that hepatocellular damage and oxidative stress caused by administration of anserine and/or Zn before or after radiation exposure are found to provide safety against γ-irradiation in rats. The results obtained do not coincide with Yukawa et al. (Yukawa et al. 1980) and Smythe et al. (SMYTHE et al. 1982) who observed that γ-irradiation caused an increase in Zn levels in various organs in the entire body. Consequently, Okada (Okada 1970) suggested that the spleen, lymph nodes, and bone marrow of the lymphoid organs are highly radiosensitive. It was clarified that Zn, released by irradiation from these damaged tissues, could accumulate in the liver, increasing its levels.
Reduced levels of Zn by both D-Gal and γ-irradiation may contribute to the zinc's ability to mitigate oxidative damage and promote the process of repair and recovery mediated by its role as important cofactors for various antioxidant enzymes such as Cu and Zn superoxide dismutase. These antioxidant enzymes are necessary to limit lipid peroxidation, help repair the modification of the oxidative base, and help restore the functionality of misfolded proteins that accompany the cellular senescence process induced by D-Gal or γ-irradiation (Marreiro et al. 2017). However, the relationship between the status of trace elements, cellular senescence, and age-related diseases is poorly studied. In addition, limited knowledge exists on the various trace element interactions, especially during cellular senescence. Cellular reactions to changes in patterns of both single trace elements and trace elements and how these (synergistic) changes affect senescent cells, organs, or the entire organism are less studied. Finally, it is important to focus on the mechanism of cellular signaling driving these changes.
In conclusion, the findings obtained in the current study showed that βOHB has the potential to minimize γ-irradiation exposure or D-Gal injection-induced hematological changes and hepatic cellular senescence in rats. At least partially, this effect can be mediated by enhancing the antioxidant function of G6PD and modulating the alteration of trace elements. These data indicate that βOHB has a protective impact against hepatic cellular senescence-related liver injury and has promising application as an antioxidant and anti-aging medication.