1. Introduction
Allantoin (5-ureidohydantoin or glyoxyldiureide) is a compound available naturally in many organisms, including microorganisms, plants, and animals [
1]. Basically, it is a degraded product of purine base bases [
2]. Many animals excrete uric acid, but some oxidize this and excrete allantoin or allantoic acid, a more water-soluble form of nitrogenous waste [
3]. Allantoin has been shown to enhance cell proliferation and collagen synthesis in human fibroblasts, which are cells that play a key role in wound healing due to their anti-inflammatory properties [
4]. In addition, allantoin is commonly used in cosmetic products due to its moisturizing and skin-soothing properties. It can help to reduce skin irritation and inflammation, as well as improve the overall texture and appearance of the skin [
5]. Allantoin influences various biological processes, particularly in wound healing and skin regeneration, making it a valuable compound for medical and cosmetic applications. Out of several plant and animal sources, crustaceans are considered good sources of allantoin, which protects them against stress, facilitates cell growth, regenerates tissues, rebuilds tissue granulation in tissue, and is responsible for slower aging [
6,
7]. The level of allantoin in most organisms depends on several physiological and environmental factors [
8]. Although allantoin is used to study metabolism [
9], as a marker for vitamins [
10], as a marker of stress [
11], atherosclerosis [
12], apoptosis studies [
13], as a signaling molecule in PI3K/Akt/GSK-3β pathway [
14], scaffold formation for medical use [
15], clinical markers, especially for chronic kidney disease [
16], for protein aggregation [
17], the variation of allantoin and its role in modulating oxidative stress physiology as a function of various sedimental and physicochemical factors of water is scantly studied in animals. The nitrogen metabolite ‘allantoin’ has multiple roles in modulating physiological activity in animals under varied abiotic factors. Still, its regulatory effect(s) on (oxidative) stress (OS) responses in organisms in general, and mud crab
S. serrata in particular, remains elusive.
Environmental temperature, humidity, and light can modulate the formation of allantoin. For example, high temperatures and low humidity can increase the formation of allantoin in some plants, while low temperatures and high humidity can decrease its formation [
18]. Similarly, allantoin production can be seasonally influenced by factors such as alkalinity, salinity, temperature, and pH, influencing the uricase mRNA level responsible for allantoin production in aquatic animals. For instance, a 7.1-fold downregulation of uricase transcript level was recorded in great spider crab
Hyas araneus in water with pH 7.54, salinity 33.5 ppt, and temperature 9 °C as compared to a control. The objective of the study was to stop the formation of allantoin in the spider crab [
19]. However, little information is available on allantoin-induced changes in invertebrates in general and under environmental fluctuations in particular. In addition to environmental factors, the formation of allantoin can also be influenced by genetic factors, as well as the presence of other compounds in the animal tissue. The altered level of allantoin has several physiological effects on organisms. Studies conducted on chronic renal failure patients have revealed that a 480% increase in allantoin affects enzymatic antioxidants activity, such as a a 96% increase in superoxide dismutase (SOD) and a 32% decrease in glutathione peroxidase (GPx) activity. In contrast to its effects on antioxidant enzymes, the lipid peroxidation (LPx) concentration was found to be 64% elevated [
20]. In another study, allantoin pretreatment reduced glutathione (GSH) depletion and restored catalase (CAT) enzyme activity in an ethanol-induced gastric ulcer model [
21]. However, free radical scavenging activity was found to be 36% lower in both
Helix aspersa and
Helix pomatia, when treated with allantoin extracted from animals, than in plants [
22]. This indicates that allantoin modulates the oxidative stress physiology of the organism, but its exact role under the varied environmental factors in any natural population is not studied.
To understand the organismal effects of allantoin under the varied physicochemical parameters of water in crustaceans such as mud crab
Scylla serrata, it is essential to identify and correlate the capacity of organisms to cope with the projected environmental changes [
23,
24,
25]. As an inhabitant of the intertidal zone,
S. serrata (mud crab) is highly influenced by environmental factors, including pH, salinity, and temperature [
26]. In this regard, the antioxidant regulatory capacity of allantoin has been noted in microbes and some invertebrates [
27,
28,
29]. Although the combined effects of various aquatic environmental factors in modulating complex cellular processes and oxidative stress responses in
S serrata have been studied, the role of allantoin in this crab under such a context is still obscure. In order to fill the knowledge gap between the potentials of allantoin and oxidative stress (physiology) in
S. serrata under changing environments, it is highly beneficial to investigate and correlate the allantoin concentration with oxidative stress parameters and environmental factors [
30]. Therefore, the hypothesis of this study was “allantoin modulates OS physiology of
S. serrata”.
4. Discussion
In this study, the allantoin was measured in two different tissues, i.e., muscle and hepatopancreas of
S. serrata as well as water physicochemical properties and sediment factors from sampling sites, which were analyzed in different seasons. Tissues such as hepatopancreas (metabolically active) and muscle (used for locomotion) were sampled to study the effects of allantoin on stress physiology. Levels of antioxidant regulatory factors such as the activity of SOD, CAT, GPx, glutathione reductase (GR) and glutathione-S-transferase, the levels of ascorbic acid (AA) and the reduced glutathione (GSH), and total antioxidant capacity in the form of DPPH inhibition capacity were quantified in the present study to draw a correlation between allantoin and the above factors [
23,
24,
25,
26,
30,
45]. Thus, the present study provides possible insights into the modulation of allantoin and its subsequent effects on the studied oxidative stress physiology parameters under the varied physicochemical parameters of the habitats. The quantification of allantoin concentration, activity of different enzymatic antioxidants, small antioxidants, and lipid peroxidation can be used to uncover its physiological effects on the organism [
46,
47,
48,
49]. Furthermore, it might be easy to use statistical tools to define the connection among potential factors that play crucial roles in modulating crab physiology in mangrove areas or in situ studies.
Being an intermediate product of the purinolytic sequence, the presence of allantoin in lower mammals is familiar but is rarely traced in marine invertebrates as they further metabolize it to ammonia and CO
2 [
50]. This is the first study on mud crabs, as well as on marine crustaceans that detects the level of allantoin and investigates its seasonal variation depending on fluctuating environmental factors. The significant impact of seasons on allantoin formation describes the role of various abiotic factors and physiological responses of tissues of mud crabs. In particular, the concentration of allantoin in hepatopancreas tissue is more relevant than in the muscle tissue of crabs, as the hepatopancreas is the central hub of metabolism. The observed allantoin level in mud crabs and its strong correlation coefficient with the physicochemical properties of water during the summer indicate that pH, temperature, and salinity collectively affect the formation of allantoin to regulate several physiological, immunological, and stress-related activities. Additionally, in previous studies, it has been shown that during drought, solar radiation, and high salinity, the accumulation of allantoin occurs in both animal and plant species. During physiological wear and tear, the allantoin modulates wound healing, cell proliferation, and inflammatory responses, which generally rise during summer as they are easily exposed to predators and dry mud [
22]. According to studies, sediment carbon content is inversely associated with urea; however, our results show a strong positive correlation with allantoin. The correlation values of Ca with allantoin are consistent with previous studies, but the reason behind such correlation is still unclear [
51]. In contrast, the correlation coefficient with Mg is opposite to the earlier findings that suggest Mg inhibits uricase, which is the primary enzyme for the conversion of uric acid to allantoin; this requires further investigation [
52]. Apart from the measured environmental factor, exercise-induced oxygen consumption is another potential explanation for the observed content of allantoin during dry seasons. Overall, environmental factors have a significant impact on allantoin formation and their subsequent role in modulating various physiological processes.
Allantoin has multiple roles in physiology and inflammatory responses, immunomodulation, and biomarkers of OS [
47,
53], so it is necessary to analyze the role of antioxidant homeostasis of the mud crab in association with allantoin. The observed TBAR level in both hepatopancreas and muscle tissues clearly indicates thermal stress during summer [
31]. In addition, the observed correlation between allantoin and LPx defines the subsequent ROS-neutralizing effects of allantoin [
54,
55,
56]. Thus, the correlation study could indirectly identify allantoin as a potent antioxidant, but this requires further study to prove it. The observed correlation values of allantoin with SOD enzyme activity suggest that the scarcity of ROS during thermal stress is due to the participation of uric acid as an antioxidant, which is evident from the increased allantoin content in tissues [
57]. However, the observed CAT activity in both hepatopancreas and muscle tissues indicates the subsequent neutralization of H
2O
2, which is the by-product of uric acid oxidation [
58]. Furthermore, the correlation coefficient values between allantoin and CAT activity support the conclusion that uric acid, rather than SOD, plays a significant role in neutralizing ROS in the present study [
55]. Apart from this, allantoin also directly upregulates the expression of SIRT1 and NRF2, which elevates CAT and SOD activity [
45,
57]. Thus, the above observation can be interpreted as the allantoin concentration being substantially related to the peroxide formation as well as a modulator of CAT and SOD activity.
According to previous studies, the elimination of peroxides from different sources like urate, amino acid oxidase, etc., is driven by glutathione systems that include GPx, GR, and GSH. The observed activity of GPx, GR, and allantoin concentration in the present study indicates peroxide formation and their neutralization. This is strongly supported by the correlation coefficient values observed between allantoin and GPx in both muscle and hepatopancreas tissues. Similarly, the r values of allantoin and GR enzymes of both tissues strongly define the presence of peroxide and its subsequent neutralization. However, the r values of GSH with allantoin do not support the same. The xenobiotics undergo detoxification through GST activity, where GSH also contributes a significant role during dry seasons, but a direct link between allantoin concentration and GST activity is still missing. However, our present correlation study strongly defines the effect of allantoin on GST activity through observed “r” values in each tissue from different sampling sites. In addition to enzymatic antioxidants, the small Ads, such as AA and GSH, of both tissues were observed to be fluctuating from the pattern of most enzymatic antioxidants, preferably due to the active participation of allantoin and uric acid-like molecules. The lack of a significant correlation between small antioxidants and allantoin further emphasizes the unsupportive role of small AD. In contrast to small AD, the observed total antioxidant capacity of both tissues was found to be significantly correlated to allantoin content, suggesting its significant role in antioxidant homeostasis.
Allantoin, considered as a potential modulator of oxidative stress physiology in a range of organisms [
56], varies seasonally in the crab tissues in the present study.
Figure 1 clearly indicates its augmented values in the summer season as compared to the other two seasons. During the summer period, all the studied environmental parameters, such as pH, temperature, salinity, organic carbon level, Ca, and Mg, were high, and the level of these parameters, along with the titer of allantoin, was low in the rainy season as compared to the other two seasons. Following the trends, all the above-studied parameters were almost observed to be at a moderate level. Such data are in agreement with the earlier observation [
24,
25,
30]. As such, the studied parameters, especially temperature and salinity, were able to raise allantoin levels in these ectothermic animals in their natural population during the summer season. As noted earlier, allantoin is metabolized to ammonia and CO
2 in invertebrates [
50]. Therefore, high salt concentrations in the hot summer season, compared to winter and rainy seasons, could be associated with the low catabolism rate of allantoin. Further detailed study is suggested on this aspect. The crab
S. serrata slowly adapts uricotelic excretory metabolism over ammonotelic metabolism as salinity increases in their environment [
34,
59]. The reverse mechanism, i.e., adaption of ammonotelic nature at low salinity observed in the rainy season, is also noted in this animal, indicating the involvement of salinity in allantoin production. This could be an adapted mechanism the crab follows to produce less ammonia at high salinity. A controlled laboratory experiment to map the allantoin level in these crabs at a constant temperature with altered salinity levels is suggested to prove this mechanism. The results confirm the rise in oxidative stress markers, such as LPx level, in the crab during summer [
24,
25,
30]. Therefore, allantoin can also be considered as one of the markers of oxidative stress in invertebrates, as already observed in vertebrates, including humans [
54,
55,
57].
Finally, a two-way ANOVA and discriminant function analysis (DFA) are used to investigate the potential role of parameters at the group or individual level. Collectively, the allantoin content and its relation between environmental factors, oxidative stress parameters, and antioxidants reveal the importance of allantoin as a participant in various physiological roles, significantly modulating antioxidant homeostasis in mud crabs. The standardized canonical coefficient values recorded individually for muscle tissue indicate that allantoin and GPx are the most reliable factors for discriminating the three groups. Similarly, for hepatopancreas tissue the canonical coefficient value suggests the importance of GR and allantoin over other factors for the discrimination of groups. These values differ when considering both tissues together, which show CAT, GR, and allantoin as the most reliable factors of discrimination among groups (
Table 6). The non-overlapping of factors is predominant when muscle and hepatopancreas tissues are taken separately. However, considering both tissues together shows a clear overlap among 4, 5, and 6, while 1 and 2 are only slightly overlapped, suggesting hepatopancreas as a significant tissue for antioxidant homeostasis over the muscle tissue (
Figure 2c). The study was conducted in nature without any control over the intrinsic or extrinsic factors of crabs; therefore, multiple such studies are suggested to overcome this limitation.
Alternatively, under the altered pH, temperature, salinity, level of organic carbon, Ca, and Mg in the habitat in the summer period, allantoin values in animals rise. In these animals, under these altered conditions of physicochemical parameters of the environment, the magnitude of oxidative damage markers such as LPx was increased, the activity of SOD was decreased, and the activity of the other antioxidant regulatory enzymes such as catalase and GPx was increased. Consequently, the level of small antioxidant-regulating enzymes and the biotransformation enzyme level was also high during the summer. It indicates the active role of the studied antioxidants in alleviating the (oxidative) stress in these animals in summer compared to the other two seasons [
60]. Therefore, the increase in the level of (oxidative) stress was accompanied by a rise in antioxidant levels (to compensate for the diminished level of SOD activity) to ameliorate the latter in animals [
55]. The elevated level of DPPH scavenging activity in the crabs at such elevated salinity and temperature is not surprising. However, the fact that it is positively correlated with allantoin levels suggests that the former plays a role in reducing stress in crabs. The antioxidant role of allantoin measured via DPPH scavenging activity in plants has clearly been noted [
61]. The chemical nature of a compound must be identical in animals and plants. So, allantoin must have ROS scavenging activity in the crabs. The role of allantoin in modulating stress primarily via NRF2 pathways is known to control several integral processes of aging, neural function, stress relieving, etc., observed in animal models [
7,
14,
29]. Furthermore, this phenomenon has been observed in plant models [
62]. However, such studies are necessary to be conducted in animals in general and specifically in ectotherms. In the present study, the adaptation of crabs to ameliorate stress via allantoin-dependent pathways is clearly noted. The rise in the allantoin level may be considered favorable to indicate its role in distressing the animals under high temperatures and salinity.