Open Veterinary Journal, (2026), Vol. 16(4):2142-2154

Research Article

10.5455/OVJ.2026.v16.i4.17

Biotransformation and detoxification enzyme (CYP1A2, CYP2E1, and GST) activities in vital organs of rats using liver extracts of Crocodylus siamensis

Thanyanant Sahabantherngsin¹, Payu Srisuporn² and Phitsanu Tulayakul^3*

¹Animal Health and Biomedical Sciences Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand

²Department of Companion Animal Clinical Medicine, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand

³Department of Veterinary Public Health, Faculty of Veterinary Medicine, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand

*Corresponding Author: Phitsanu Tulayakul. Department of Veterinary Public Health, Faculty of Veterinary Medicine, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand. Email: fvetpnt [at] ku.ac.th

Submitted: 17/11/2025 Revised: 11/03/2026 Accepted: 24/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Detoxification enzymes, including cytochrome P450 isoforms (CYP1A2 and CYP2E1) and glutathione-S-transferase (GST), play key roles in xenobiotic metabolism and organ protection. Although crocodile-derived bioactive compounds have gained biomedical interest, the in vivo effects of Crocodylus siamensis liver extract on mammalian detoxification pathways remain largely unexplored.

Aim: To evaluate the effects of oral administration of C. siamensis liver extract on phase I (CYP1A2, CYP2E1) and phase II GST detoxification enzyme activities, kinetic parameters (V_max, K_m, catalytic efficiency), and selected blood biochemical profiles in rat liver and kidneys.

Methods: Male Wistar rats (n=5) were orally administered freeze-dried C. siamensis liver extract at 0, 50, 100, 200, 400, or 800 mg/kg for 7 days. Liver and kidney tissues were collected for microsomal and cytosolic fractionation. Enzyme activities were quantified using specific substrates (3-cyano-7-ethoxy coumarin for CYP1A2, 7-methoxy-4-trifluoromethylcoumarin for CYP2E1, and 1-chloro-2,4-dinitrobenzene for GST), and kinetic parameters were calculated by Michaelis–Menten modelling. Serum albumin, total protein, cholesterol, and glucose were analyzed using automated clinical assays. Data were assessed by one-way analysis of variance with Tukey’s post hoc test for variables that met parametric assumptions, whereas non-parametric data were analyzed using the Kruskal–Wallis test.

Results: Crocodile liver extract produced dose-dependent and tissue-specific modulation of detoxification enzymes. Hepatic CYP1A2 showed a biphasic response, with catalytic efficiency increasing markedly at 400–800 mg/kg. Hepatic CYP2E1 activity increased toward 400 mg/kg before stabilizing. In the kidneys, CYP1A2, CYP2E1, and GST activities were consistently elevated, with GST showing the strongest induction; V_max increased from 0.16 to 0.59 µmol/minute/mg at 800 mg/kg. Several groups exhibited enhanced catalytic efficiency despite reduced V_max, indicating improved substrate affinity. Blood analyses showed increased serum albumin and total protein, while cholesterol and glucose were reduced, suggesting metabolic benefits associated with liver-related biochemical changes.

Conclusion: Oral supplementation with C. siamensis liver extract modulates phase I and phase II detoxification pathways in rats, with pronounced induction in renal tissues and high-dose enhancement in hepatic enzymes. These findings provide the first in vivo evidence that reptile-derived liver extract can enhance xenobiotic metabolism and support metabolic homeostasis, highlighting its potential as a natural bioactive supplement in veterinary applications.

Keywords: Crocodylus siamensis, Detoxification enzymes, CYP, GST, Biotransformation.

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Introduction

The liver and kidneys are the primary organs responsible for xenobiotic metabolism and elimination of xenobiotics through enzymatic processes classified as phase I and phase II reactions. Phase I enzymes, particularly cytochrome P450 (CYP450) isoforms such as CYP1A2 and CYP2E1, are involved in the oxidation and activation of lipophilic compounds, whereas phase II enzymes, such as glutathione-S-transferases (GSTs), facilitate the conjugation with glutathione, promoting its excretion and reducing its toxicity (Hayes and Pulford, 1995; Thiendedsakul et al., 2020; Srisuksai et al., 2023). The coordinated activity of these enzymes plays a key role in maintaining cellular homeostasis and defending tissues from chemical insults (Bradford, 1976; Singh and Reindl, 2021). Species- and tissue-specific differences in these detoxification systems provide valuable insights into comparative physiology and toxicological responses.

Crocodylus siamensis (Siamese crocodile), a freshwater crocodilian species endemic to Southeast Asia, is currently listed as a critically endangered species. However, successful domestication and farming, particularly in Thailand, have enabled sustainable use of leather and meat products (Lapbenjakul et al., 2017; Ariyaraphong et al., 2023). In recent years, the biomedical value of crocodile-derived by-products, especially the liver and internal organs, which are rich in bioactive compounds but are often discarded as waste or used in aquaculture, has attracted considerable attention (Santativongchai et al., 2020; Srisuksai et al., 2023).

Earlier research indicates that crocodile oil is rich in lipids and fatty acids with antioxidant, anti-inflammatory, and wound-healing properties (Parunyakul et al., 2023). In contrast, the present study used freeze-dried crocodile liver powder, which preserves not only lipid fractions but also proteinaceous and water-soluble components. This preparation provides a broader biochemical context, particularly regarding enzymatic cofactors and detoxification-related proteins that are absent in oil-based extracts (Santativongchai et al., 2022). Using whole liver powder, we aimed to extend current knowledge beyond lipid-derived effects and explore the potential contributions of non-lipid bioactive compounds.

Liver powder contains not only lipids but also proteins, enzymatic cofactors, and water-soluble antioxidants that are absent in oil-based extracts. Despite promising in vitro results, evidence of the physiological impact of crocodile liver extract in vivo remains limited, particularly regarding organ-specific detoxification enzyme activity. Evaluating these effects in the liver and kidneys of model animals can provide mechanistic insights into xenobiotic metabolism and help bridge findings from cell studies to whole-organism physiology. Such data are crucial for assessing the safety and potential therapeutic applications of C. siamensis liver extract in veterinary pharmacology and toxicology.

The antioxidant, anti-inflammatory, and hepatoprotective effects of crocodile liver components have been highlighted in previous studies. In vitro investigations demonstrated that C. siamensis liver extract reduced oxidative stress and apoptosis in HepG2 cells, potentially by modulating detoxification enzymes (Thiendedsakul et al., 2022). Crocodile hepatocytes and tissues have also been shown to express active GSTs and Cytochrome P450 enzymes with distinct distribution profiles (Tassaneeyakul et al., 1994; Thiendedsakul et al., 2020; Ariyaraphong et al., 2023); however, in vivo evidence regarding the physiological effects of crocodile liver extract on xenobiotic metabolism remains limited. The present study aimed to investigate the effects of crocodile liver extract on xenobiotic metabolism. Systematic evaluation of enzyme activity in multiple organs, such as the liver and kidneys, remains underexplored.

Detoxification enzymes in companion animals, such as cats and dogs, have gained increasing attention as relevant models for veterinary pharmacology and toxicology. Phase I and II enzymes in these species show considerable interspecies differences compared with those in humans, significantly influencing drug metabolism and toxicity (Ismail et al., 2021). In addition, a study on inductive substances on phase 2 enzyme activity in rats reported that sulforaphane significantly induced nicotinamide quinone oxidoreductase or total GST enzyme activity in the liver, kidney, and bladder tissues. Moreover, rat liver contains enzymes, including cytochrome P450, flavin-containing monooxygenase, alcohol dehydrogenase , aldehyde dehydrogenase, esterase, UDP-glucuronosyltransferase, and GST, but lower activities in placental tissue (Jones & Brooks, 2006; Fabian et al., 2016).

As phase I enzymes, cytochrome P450 isoforms remain central to hepatic and renal metabolism. (Lootens et al., 2022) elucidated the Michaelis-Menten kinetics of CYP1A2 and CYP3A4 in pooled human liver microsomes using aflatoxin B1 as a model substrate to provide a comparative baseline for interspecies metabolism, while (Uno et al., 2024) investigated CYP4A activity across multiple species, including cats, dogs, pigs, and humans, with results revealing striking differences in expression and catalytic efficiency. This interspecies variability in CYP-mediated oxidation further reinforces the need for cautious extrapolation when applying findings from one species to another. The results of these studies underscore the functional diversity of detoxification systems across mammalian species and support the rationale for investigating the modulatory potential of reptile-derived extracts in traditional and non-traditional animal models. Understanding organ- and species-specific enzyme responses is essential for assessing the safety and efficacy of natural products such as crocodile liver extract. This study examined the effect of C. siamensis liver extract on CYP1A2, CYP2E1, and GST activities in the liver and kidneys of rats as a foundational step for potential application in veterinary contexts.

The effects of oral administration of C. siamensis liver extract on CYP1A2, CYP2E1, and GST activity in rat liver and kidneys were examined. The kinetic parameters (K_m and V_max) were also evaluated to determine organ-specific responses to enzymatic modulation. Our findings offer novel insights into the physiological modulation of biotransformation systems by reptile-derived natural extracts and contribute to comparative toxicology and pharmacology. In addition, this study also investigated blood-chemical parameters, including albumin, total protein, cholesterol, and glucose, which provide an overview of liver function and metabolic responses in rats treated with crocodile liver extracts. Enzyme kinetic parameters (K_m and V_max) and blood biochemical profiles were also evaluated to provide fundamental insights for potential veterinary applications. These investigations are particularly focused on detoxification capacity in companion animals to reduce xenobiotic risks. Therefore, this study aimed to examine the effects of C. siamensis liver extracts on the activities of phase I (CYP1A2, CYP2E1) and phase II (GST) detoxification enzymes in rat liver and kidney tissues.

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Materials and Methods

Animals and treatment

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Kasetsart University (Approval ID: ACKU61-VET-002; Approval date: January 23, 2018) and conducted in accordance with the ethical guidelines for the care and use of laboratory animals.

Male Wistar rats (24 weeks old) were obtained from The Nomura Siam International Co., Ltd. (Thailand) and M-CLEA Bioresource Co., Ltd. (Thailand). The animal organs operated in this study were previously used by Thiendedsakul et al. (2022). The rats were housed under controlled environmental conditions (22°C ± 2°C, 55% ± 5% humidity, 12:12 hours light-dark cycle) with free access to food and water. All animals were clinically healthy at the start of the study, as confirmed by veterinary examination. Only male Wistar rats (24 weeks of age) were selected to minimize variability from sex-related hormonal influences and to represent a young adult stage with stable metabolic activity. Oral administration was implemented by gavage once daily to ensure accurate dosing. Rats were monitored daily for food and water consumption, clinical signs of toxicity, and behavioral changes throughout the study. No abnormal clinical signs, changes in feed intake, or mortality were observed in any group, even at the highest dose tested (800 mg/kg). After 1 week of acclimatization, the rats were randomly assigned into six groups (n=5 per group) and orally administered C. siamensis liver extract at doses of 0 (control), 50, 100, 200, 400, and 800 mg/kg body weight once daily for 7 consecutive days (Fig. 1).

Fig. 1. Experimental design showing treatment groups of male Wistar rats receiving 0–800 mg/kg of C. siamensis liver extract for 7 days, with subsequent collection of liver and kidney tissues for microsomal (CYP1A2, CYP2E1) and cytosolic (GST) enzyme assays.

Preparation of the liver extract

Mixed sexes of C. siamensis liver at the age of 3 years were obtained from a certified crocodile farm in Thailand. The liver was washed, chopped into small pieces, immediately frozen at −80°C, and finely ground into a homogeneous substance. The extract was prepared by dissolving the liver powder in distilled water, followed by sonication and centrifugation. The supernatant was filtered and stored at −20°C until further use. The extraction procedure was adapted from the method described by Thiendedsakul et al. (2022). The freeze-dried crocodile liver powder used in this study differs from crocodile oil preparations described in previous reports, as it retains proteinaceous and water-soluble constituents in addition to lipids (Santativongchai et al., 2020; Santativongchai et al., 2022).

Collection and fractionation of cytosol and microsomes

Twenty-four hours after the final dose, the rats were euthanasia by CO₂ inhalation. Liver and kidney tissue samples were immediately collected, rinsed with ice-cold phosphate-buffered saline, blotted dry, and snap frozen in liquid nitrogen. The cytosolic and microsomal fractions were preparing by differential centrifugation according to the method described by Thiendedsakul et al. (2022). Briefly, the tissues were homogenized in an ice-cold buffer containing protease inhibitors and then centrifuged at 10,000 g for 15 minutes at 4°C. The resulting supernatant was centrifuged at 100,000 g for 60 minutes at 4°C to separate the cytosolic and microsomal fractions. The protein concentrations of each fraction were quantified using the Bradford assay (Bradford, 1976).

Blood collection and biochemical analysis

After euthanasia, blood samples were collected directly from the heart using serum clot activator tubes for biochemical analysis. The assays included the determination of serum albumin, total protein, cholesterol, and glucose levels. These assays were performed using an automated clinical chemistry analyzer according to the manufacturer’s instructions.

Enzyme activity assays

Glutathione-S-transferase activity was measured based on the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene (CDNB), and the absorbance was recorded at 340 nm following the manufacturer’s protocol (Habig et al., 1974; Bradford, 1976; Thiendedsakul et al., 2020). The CYP1A2 activity was assessed using 3-cyano-7-ethoxy coumarin as a specific substrate, and the metabolite product fluorescence was measured (Tassaneeyakul et al., 1994;Miller et al., 2000). CYP2E1 activity was determined using 7-methoxy-4-trifluoromethylcoumarin (7-MFC) as a substrate (Stresser et al., 2002; Thiendedsakul et al., 2022), and its hydroxylated product was fluorescently detected. All enzyme assays were performed in triplicate in 96-well microplates. The kinetic parameters (V_max and K_m) were obtained by fitting the data to the Michaelis-Menten equation using nonlinear regression analysis (Segel, 1975).

Statistical analysis of the data

Data are presented as mean ± standard deviation (SD). Data distribution was assessed before statistical analysis. For variables that did not meet the assumptions of the parametric tests, the Kruskal–Wallis test was used for group comparisons. For variables that met the assumptions of parametric analysis, differences among groups were evaluated using one-way analysis of variance followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant. GraphPad Prism version 10.3 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. The catalytic efficiency (V_max/K_m) was emphasized as an indicator of enzyme performance and is presented to support comparisons among treatment groups.

Ethical approval

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Kasetsart University (Approval ID: ACKU61-VET-002; Approval date: January 23, 2018) and were conducted in accordance with the ethical guidelines for the care and use of laboratory animals.

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Results

The male Wistar rats (24 weeks old, weighing 200–250 g) were housed under con-trolled environmental conditions (22°C ± 2°C, 12 hours light/dark cycle) with free access to standard laboratory chow and water. After a 7 days acclimation period, the animals were orally administered C. siamensis liver extract once daily for seven consecutive days. The liver extract was prepared by homogenizing fresh crocodile liver in cold phosphate buffer, followed by solvent extraction and lyophilization to preserve the bioactive components (Thiendedsakul et al., 2022). Care was taken during the extraction process to maintain the integrity of the compounds potentially responsible for modulating detoxification enzymes. Following treatment, the enzymatic activities in the liver and kidney tissues of the rats were analyzed. Overall, oral administration of C. siamensis liver extract significantly modulated detoxification enzyme activities in a dose- and tissue-dependent manner. Both hepatic and renal tissues exhibited measurable alterations in phase I and II enzyme function, with clear evidence of catalytic efficiency changes. These general trends are summarized below, followed by a detailed description of each enzyme.

CYP1A2 activity

CYP1A2 activity in the liver showed a biphasic pattern. The control group displayed the highest V_max (1.3 × 10⁵ µmol/minute/mg protein), which decreased by 50–200 mg/kg but returned to a comparable level at 400–800 mg/kg. The catalytic efficiency peaked at 400 and 800 mg/kg (1.4 × 10⁴ ml/minute/mg protein), suggesting that CYP1A2 enhanced catalytic efficiency (V_max/K_m ratio) at high doses but not at lower doses (Fig. 2B). In the kidney, CYP1A2 activity was significantly induced by all doses compared with that in the control group, with the highest catalytic efficiency found at 800 mg/kg (8.5 × 10¹ ml/minute/mg protein), as shown in Figure 2A and B.

CYP2E1 activity

In the liver, CYP2E1 activity increased in a dose-dependent manner, reaching a peak V_max of 1.0 × 10⁴ µmol/minute/mg protein at 400 mg/kg, with a corresponding catalytic efficiency of 1.1 × 10⁻³ (Fig. 2C and D). In the kidney, CYP2E1 activity was significantly induced at 100 and 200 mg/kg, with the highest catalytic efficiency of 8.3 × 10² ml/minute/mg protein observed at an 800 mg/kg dose, despite a decline in V_max to 1.4 × 10³ µmol/minute/mg protein. Although the maximal reaction velocity (V_max) decreased at the highest dose, the affinity of the enzyme for its substrate (K_m) increased catalytic efficiency. This modulation indicated a shift toward more efficient substrate processing at lower enzyme concentrations or altered enzyme kinetics in the kidney than in the liver.

Fig. 2. Enzyme activities of CYP1A2, CYP2E1, and glutathione S-transferase (GST) in the liver and kidney tissues of rats after oral administration of crocodile liver extracts at different doses. (A) CYP1A2 activity in rat liver, (B) CYP1A2 activity in rat kidney, (C) CYP2E1 activity in rat liver, (D) CYP2E1 activity in rat kidney, (E) GST activity in rat liver, and (F) GST activity in rat kidney. Data are expressed as mean ± SD (n=5). Hash symbols (#) indicate significant differences compared to the control group (p < 0.05). Asterisks (*) indicate significant differences compared to the other groups (*p < 0.05, **p < 0.01, ***p < 0.001), as determined by one-way ANOVA followed by Tukey's post hoc test (p < 0.05).

GST universal activity

In the liver, the V_max of GST activity decreased at low doses (50–100 mg/kg), followed by an increasing trend at 200–800 mg/kg. The highest activity was observed at 400 mg/kg (0.90 µmol/minute/mg protein), while the catalytic efficiency remained stable (Fig. 2F). In the kidney, GST activity increased at doses of 50, 400, and 800 mg/kg, with the maximal V_max observed at 800 mg/kg (0.50 µmol/minute/mg protein), resulting in increased catalytic efficiency of 1.32 ml/minute/mg protein observed at 800 mg/kg (Fig. 2E and F). These results concurred with the graphical representation in Table 1, demonstrating the dose-dependent modulation of enzyme activity across the three enzymes (GST, CYP1A2, and CYP2E1) in liver and kidney tissues.

Table 1. Kinetic parameters of CYP450 and GST activities in rat liver and kidney tissue (n=5).

Remarks: The kinetic parameters, V_max and K_m, for CYP1A2, CYP2E1, and GST activities were determined by Michaelis-Menten kinetics using various substrate concentrations for each treatment group in rat liver and kidney tissues. The V_max/K_m ratio represents the catalytic efficiency. Data were derived from Michaelis-Menten curve fits for each group; "nd" indicates that the parameter could not be determined. Superscript letters (a, b) indicate groups that differ significantly among treatments (p < 0.05, Kruskal–Wallis test). The groups without superscripts did not differ significantly.

Michaelis-menten kinetics of xenobiotic metabolizing enzymes

The Michaelis–Menten kinetics for CYP1A2, CYP2E1, and GST activities in rat liver and kidney tissue after oral administration of crocodile liver extracts are shown in Fig. 3. The relationships between substrate concentration and reaction velocity for each enzyme and treatment group are shown in the plots.

For CYP1A2, the Michaelis–Menten curves in the liver (Fig. 3A) revealed that all treatment groups, including the control, exhibited typical hyperbolic saturation kinetics, reaching maximal velocity with increasing substrate concentration. The curves for doses of 200 mg/kg and 400 mg/kg showed higher maximal velocities compared with the control and the other treatment doses, but with no significant differences. The curves for the kidney (Fig. 3B) demonstrated dose-dependent effects. The 50 and 100 mg/kg doses resulted in lower maximal velocities compared with the control group. In contrast, the 200, 400, and 800 mg/kg doses revealed higher maximal velocities, indicating a rapid induction of CYP1A2 activity in the renal organ.

For CYP2E1, the Michaelis–Menten plots for the liver samples (Fig. 3C) indicated that all groups displayed saturation kinetics with increasing 7-MFC concentration. No substantial differences in the maximal velocities were recorded between the control and treatment groups. For the kidney (Fig. 3D), the 50 mg/kg dose showed a noticeable increase in the maximal velocity compared with the control group, revealing the induction of renal CYP2E1 activity. The curves for the other doses remained similar to, or slightly above, the control group, with no statistically significant alterations.

Fig. 3. Michaelis–Menten kinetics of CYP1A2, CYP2E1, and GST in liver and kidney tissue across different treatment doses Panels: (A) CYP1A2 liver, (B) CYP1A2 kidney, (C) CYP2E1 liver, (D) CYP2E1 kidney, (E) GST liver, and (F) GST kidney. The kinetic parameters were derived from nonlinear regression fits at multiple substrate concentrations.

Table 2. Blood-chemical parameters of rats treated with Crocodylus siamensis liver extract for 7 days (mean ± SD, n=5). Cholesterol and glucose levels decreased, whereas albumin and total protein levels increased significantly compared with the control group (p < 0.05).

Fig. 4. Blood biochemical profiles of rats treated with different doses of the extract Panels: (A) albumin, (B) total protein, (C) cholesterol, and (D) glucose. Data are mean ± SD (n=5). # indicate p < 0.05 versus control; * indicates differences among groups (ANOVA, Tukey’s test).

The Michaelis–Menten curves of the GST activity in the liver (Fig. 3E) showed that the reaction velocity increased with the CDNB concentration in all groups. The curves overlapped, indicating that the crocodile liver extracts did not substantially alter the overall kinetic profile of hepatic GST activity across the tested doses. In contrast, the GST activity in the kidney (Fig. 3F) exhibited a dose-dependent increase in GST activity. Curves for the 50, 100, 200, 400, and 800 mg/kg treatment groups progressively showed higher velocities compared with the control group, whereas the 400 and 800 mg/kg doses displayed the highest reaction rates. This evidence strongly supports the induction of renal GST activity by liver extracts from crocodiles. The five measurements applied at each substrate concentration for the control and treatment groups provided the baseline calculation of the kinetic parameters (V_max and K_m), as shown in Table 1.

Blood-chemical parameters

Blood-chemical analysis revealed that supplementation with crocodile liver extract significantly increased serum albumin and total protein levels compared with the control group, while cholesterol and glucose levels were decreased. These findings indicated an improvement in protein balance and a potential reduction in metabolic risk factors in rats (Table 2, Fig. 4).

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Discussion

This study provided direct in vivo evidence that the oral administration of C. siamensis liver extract significantly modulated detoxification enzyme activities in a dose- and tissue-specific manner. The findings from the rat liver and kidneys indicated that the bioactive compounds within the extract were orally bioavailable and exerted biotransformation effects in metabolically active tissues. Comparable modulations of hepatic and renal detoxification pathways were previously demonstrated for phytochemicals such as curcumin, silymarin, and green tea catechins, which primarily regulate phase I and II enzymes through nuclear receptor activation and re-dox-sensitive signalling pathways (Hakkola et al., 2020). This study provided the first in vivo evidence that an animal-derived extract, specifically from C. siamensis liver, exhibited comparable or potentially superior enzymatic modulation, as evidenced by significant dose-dependent changes in CYP1A2, CYP2E1, and GST activities, along with enhanced catalytic efficiency in the hepatic and renal tissues. In addition, supplementation with crocodile liver extracts could enhance total protein and albumin levels while decreasing cholesterol and glucose levels. It was quite a benefit for animals after receiving this supplement because it contains rich of crude protein. However, further study should focus on the mechanism of alleviation of cholesterol and glucose levels. Although rat receiving freeze-dried crocodile blood could reduce blood glucose via insulin-like growth factor-1, this supplement is supposed to be a good nutritious medical value containing similar active ingredients (Siruntawineti et al., 2013).

After 7 days of supplementation, significant alterations in the phase I enzymes CYP1A2 and CYP2E1, as well as in the phase II enzyme GST, were observed. CYP1A2, a key enzyme responsible for the oxidative metabolism of a wide range of xenobiotics and procarcinogens such as heterocyclic amines and caffeine, exhibited like biphasic hepatic response (suppression at 50–200 mg/kg; induction at 400–800 mg/kg where statistically significant; Table 1), rather than a classical monotonic dose-dependent relationship. This biphasic pattern suggested a threshold-dependent or hermetic mechanism, consistent with findings reported for several phytochemicals that show non-monotonic dose-response relationship. Such effects are often mediated by nuclear receptor pathways, particularly the aryl hydrocarbon receptor and constitutive androstane receptor, which coordinately regulate xenobiotic-metabolizing enzymes including CYP1A2 and CYP2E1 (Köhle and Bock, 2009; Goedtke et al., 2021). Similarly, plant-derived polyphenols such as curcumin and green-tea catechins have been shown to differentially modulate CYP1A2 activity in vivo through receptor-mediated transcriptional mechanisms (Thapliyal and Maru, 2001; Wang et al., 2022). The catalytic efficiency of CYP1A2 increased at higher doses, indicating enhanced enzyme functionality, which aligns with adaptive metabolic responses observed in dose-dependent xenobiotic detoxification (Low et al., 2024).

Hepatic CYP2E1 activity demonstrated a dose-dependent increase, peaking at 400 mg/kg. CYP2E1 plays a pivotal role in the metabolism of low-molecular-weight toxicants, such as ethanol, acetaminophen, benzene, and nitrosamines, and is a known source of reactive oxygen species (ROS) during substrate turnover (Bammler et al., 2000). Despite a decrease in V_max at the highest dose (800 mg/kg), the catalytic efficiency remained elevated and enhanced substrate affinity. These findings implied that C. siamensis liver extract bolstered the capacity of rat liver to detoxify specific environmental and drug-related toxicants, aligning with previous reports on dietary modulators of CYP activity. However, the induction of Phase I enzymes should be interpreted cautiously because the increased CYP activity may also lead to either the formation of reactive metabolites or health promotion, as expected.

GSTs are vital phase II enzymes that catalyse the conjugation of glutathione to electrophilic and toxic compounds, facilitating their elimination. GSTs are also implicated in cancer susceptibility and chemoresistance, with polymorphisms and differential expression of iso-enzymes influencing cellular oxidative stress responses (Bammler et al., 2000; Singh & Reindl, 2021a). In this study, hepatic GST activity was reduced at low doses but significantly increased at doses ≥200 mg/kg, reflecting enhanced phase II detoxification potential. These results corroborate previous in vitro findings where C. siamensis liver extract reduced intracellular ROS and lipid peroxidation while upregulating endogenous antioxidants such as superoxide dismutase and catalase in HepG2 cells, leading to decreased apoptosis and improved cell viability (Thiendedsakul et al., 2022). Moreover, the pi class in the crocodile liver was protected from mutagenic and carcinogenic activities compared with other species, and GST could metabolize toxic electrophiles and conjugate them to more easily excrete them into bile or urine. (Whalen and Boyer, 1998).

Renal tissues showed heightened sensitivity to the liver extract, while GST activity was significantly elevated, even at the lowest dose tested (50 mg/kg), with CYP1A2 and CYP2E1 catalytic efficiencies peaking at 800 mg/kg. These observations indicated that oral liver extract supplementation enhanced hepatic and renal xenobiotic metabolism, consistent with the previously reported renal protective effects of bioactives (Santativongchai et al., 2022; Fungfuang et al., 2023; Putra et al., 2023). The renal benefits arose from increased conjugation of nephrotoxic compounds via GST, diminished oxidative stress through antioxidant pathway upregulation, and improved clearance of ROS-generating substrates.

In parallel with enzymatic modulation, the observed blood biochemical changes further support the systemic metabolic effects of C. siamensis liver extracts in mammals, such as rats. Increasing serum albumin and total protein levels indicated that hepatic function was enhanced, and proteins could be beneficial. The reductions in cholesterol and glucose suggested a beneficial modulation of lipid and carbohydrate metabolism, aligning with the previously reported metabolic regulatory effects of reptile-derived products and crocodile oil in experimental animals (Santativongchai et al., 2022; Thiendedsakul et al., 2020). Moreover, the results showed enhanced CYP and GST activities, implying that oral liver extract supplementation not only improved phase I and II detoxification capacity but also positively influenced systemic metabolic homeostasis.

Maximal enzymatic responses occurred at higher doses with partial activation at lower doses, suggesting nonlinear dose-response relationships possibly due to differences in compound bioavailability, absorption, or tissue-specific distribution. This finding underscored the importance of dose optimization to maximize therapeutic efficacy while minimizing metabolic burden. These findings demonstrate clear modulation, with statistically significant induction observed at 400–800 mg/kg (p < 0.05), highlighting the importance of optimizing dose ranges for therapeutic efficiency for CYP1A2 in the liver rather than kidney. Moreover, the modulation of GST could be induced in the liver of rat receiving Crocodile liver extract started from 100 to 800 mg/kg, compared with the control group but not significant in the kidney sample. Thus, it was quite ascertained that the crocodile liver extracts could modulated liver enzyme activities rather than kidney, especially for CYP1A2 and GST activities. Organ differences towards major enzyme activities could be different among organs, which used to be reported in the rat (Jones & Brooks, 2006; Fabian et al., 2016). A key strength of this study lies in the detailed kinetic characterization of enzyme activity, including V_max, K_m, and catalytic efficiency. However, some relatively low r² values observed for kidney CYP1A2 and CYP2E1 (Table 1) may be attributed to biological variability in renal enzyme expression and the limited number of experimental animals, which could affect the curve fitting of Michaelis–Menten kinetics. Such quantitative enzymatic profiling for animal-derived extracts is scarce, and results provide valuable mechanistic insights into metabolic modulation across tissues and dosage ranges (Thiendedsakul et al., 2022; Srisuksai et al., 2023).

Detoxification enzymes, such as CYP1A2, CYP2E, and GST, exhibit interspecies variation, especially in companion animals, such as cats and dogs. Dogs express active GSTP1-1 isoforms critical for xenobiotic conjugation but may harbor breed-specific polymorphisms, increasing the risk of drug toxicity (Sacco et al., 2017; Ismail et al., 2021; Fedets et al., 2023). Cats display comparatively limited CYP450 isoform expression, particularly CYP2E and CYP4A, reducing their xenobiotic metabolism capacity (Hoshino et al., 2024). These differences highlight the need for species-specific evaluation when applying natural enzyme modulators, such as C. siamensis liver extract, in veterinary medicine. GST enzymes have been implicated in cancer chemoprevention and therapy because of their roles in detoxifying carcinogens and modulating oxidative stress (Singh and Reindl, 2021b). Plant-derived natural compounds dominate detoxification research, but animal-derived bioactive compounds, such as crocodile products, are now emerging as potent underexplored resources. Crocodile blood and bile exhibit antimicrobial, hepatoprotective, and immunomodulatory properties; however, mechanistic and kinetic evaluations are limited. This study highlighted the distinct advantage of crocodile liver extract in the dual-phase enzymatic enhancement of CYP450 and GST systems with measurable kinetic benefits. Crocodile liver is rich in xenobiotic-metabolizing enzymes and cofactors, providing synergistic effects and conferring superior bioactivity compared with other crocodile-derived components and plant extracts. However, this study had some limitations, including the short duration of supplementation and the absence of molecular-level analyses, such as gene expression or protein quantification. To fully elucidate the therapeutic potential of crocodile liver extracts, future studies should explore their long-term effects, pharmacokinetics, and molecular mechanisms.

Oral administration of C. siamensis liver extracts induced systemic modulation of key xenobiotic-metabolizing enzymes, enhancing liver and kidney detoxification capacities in phases I and II. This modulation was dose-dependent, with the most pronounced effects observed at higher doses. These findings support the potential of crocodile liver extract as a natural modulator of detoxification pathways, with promising applications in veterinary therapeutics and biomedical research, particularly in contexts involving oxidative stress and cancer prevention.

Importantly, the results obtained in rats could have beneficial implications for other animals, such as dogs and cats (Court, 2013; Robinson, 2023). In addition, cats exhibit a deficiency for glucuronidation pathways and reduced CYP450 activity, thereby predisposing them to adverse drug reactions. The observed induction of CYP1A2, CYP2E1, and GST by C. siamensis liver extracts that supplementation could enhance detoxification capacity and reduce the risk of xenobiotic accumulation in these species. In veterinary practice, these products could be an alternative choice for health support in companion animals. These perspectives reinforce the relevance of the findings beyond experimental rodents and highlight the need for further species-specific validation studies.

This study has some limitations. The histopathological assessment of liver and kidney tissue was excluded, nor was the oxidative stress studied, which might be carried on in further studies. The histopathological examination of liver tissue in the rat has been examined but not yet for the kidney; however, there was no significant difference in abnormal findings between the treatment groups. Exceptional found for hepatic steatosis was higher in the highest doses compared with the control group. It could be presumed that fat accumulation declined after treatment with crocodile liver extracts (data not shown). Nevertheless, no mortality, abnormal clinical signs, or adverse serum biochemistry was observed, even at the highest dose. Therefore, future studies should incorporate detailed histopathological and toxicity evaluations, validate tissue safety, and refine dose justification for clinical applications in other animal species, such as dogs and cats.

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Conclusion

Oral administration of liver extracts of C. siamensis modulated phase I (CYP1A2, CYP2E1) and phase II (GST) detoxification enzymes in rat liver and kidneys in a dose- and tissue-dependent manner. Hepatic CYP enzymes exhibited strong induction at higher doses (400–800 mg/kg), indicating either enhanced oxidation-reduction metabolism or reactive metabolites, while renal enzymes showed consistent activation across all doses, reflecting greater tissue sensitivity. Renal GST activity increased significantly even at the lowest dose (50 mg/kg), early engagement of conjugation pathways.

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Acknowledgments

The authors are grateful to the Faculty of Veterinary Medicine, Kasetsart University, Thailand.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study was funded by the Faculty of Veterinary Medicine, Kasetsart University, Thailand.

Authors' contributions

Thanyanant Sahabantherngsin: conducted the experiments, collected and analyzed the data, and drafted the manuscript. Payu Srisuporn: contributed to the study design, supervised the laboratory procedures, and provided critical revisions to the manuscript. Phitsanu Tulayakul: conceived and designed the study, supervised the overall project, and finalized the manuscript as the corresponding author. All authors have read and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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