Hormetic association between sodium nitrate and liver enzymes: a study in female and male rats

Abstract

This study aims to evaluate the long-term dose-dependent effects of sodium nitrate on serum ALT, AST, and ALP in healthy female and male rats. A total of 120 rats (60 females and 60 males) were divided into 6 subgroups (n = 10). In each sex, a control group received regular tap water, while five treatment groups received sodium nitrate in tap water (50, 100, 150, 250, and 500 mg/L). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and nitric oxide (NO) metabolites (NOx) were measured at baseline (month 0) and after 6 mo. Hormetic zones for sodium nitrate in female rats were 10–230 mg/L for ALT, 20–180 mg/L for AST, and 8–270 mg/L for ALP. In males, the zones were 5–190 mg/L for ALT, 7–180 mg/L for AST, and 7–265 mg/L for ALP. Serum NOx levels were negatively correlated with ALT (r = −0.498) and AST (r = −0.320) at moderate doses (100–150 mg/dL) but positively correlated with ALT (r = 0.500) and AST (r = 0.300) at higher doses (250–500 mg/dL (all P < 0.05). Sodium nitrate exhibited a J-shaped dose–response relationship with liver function tests (LFTs) in both female and male rats. Protective effects were observed at moderate doses (100 mg/L for ALT and AST; 150 mg/L for ALP), while low doses (<10 mg/L) were ineffective, and high doses (>200 mg/L for ALT/AST; >250 mg/L for ALP) were toxic. These findings highlight the dual potential of sodium nitrate as both beneficial and harmful, depending on the dosage in healthy state.

Introduction

Abnormal liver function has a global prevalence of 9–22%^1,2 contributing to approximately two million deaths annually.³ Elevated serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are key markers of liver dysfunction, along with alkaline phosphatase (ALP), a less liver-specific enzyme, are associated with increased risks of type 2 diabetes,^4,5 cardiovascular disease,^6,7 and overall mortality.⁸ Even within normal ranges, higher levels of ALT, AST, and ALP levels are linked to an elevated risk of developing metabolic syndrome and cardiovascular disease, as well as increased mortality. For instance, healthy adults with serum ALT levels in the upper quartiles of the reference range are at a significantly greater risk of metabolic syndrome, with 1.8-fold,⁹ 4.4-fold,¹⁰ and 7.9-fold¹¹ increased risks reported in separate studies. Similarly, for AST values of 20–29, 30–39, 40–49, and 50–99 U/L (compared to <20 U/L), the adjusted risk ratios for all-cause mortality in adult women and men have been reported as 1.3, 1.7, 2.7, and 5.4-fold, respectively.¹² Elevated ALP levels are also predictive of cardiovascular mortality, with individuals in the third and fourth quartiles having 33% and 39% higher risks, respectively, compared to those in the first quartile.¹³

Nitric oxide (NO), a critical signaling molecule, is synthesized through two pathways: the classical L-arginine-NO pathway, in which NO is synthesized by NO synthase (NOS) enzymes, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), and the nitrate-nitrite-NO pathway.¹⁴ Evidence stongly suggests that reduced hepatic NO levels lead to elevations in ALT, AST, and ALP,¹⁵ while decreased eNOS activity is associated with liver diseases in both humans¹⁶ and animals models.^17,18 Physiological concentrations of eNOS-derived NO are protective, whereas pathological concentrations of iNOS-derived NO have detrimental effects on liver function.¹⁹

While the effect of nitrate, a NO donor, on ALT, AST, and ALP in healthy animals have been investigated, prior studies have primarily been conducted on male rats^20–25 and mice.¹⁵ These studies focused on short-term nitrate administration to rats, ranging from 10²² to 60²⁵ days, typically using a single dose,^15,21,23,24 with only few studies employing two²⁵ or three doses.^20,22 Nitrate concentrations in these studies ranged from 40 mg/L¹⁵ to as high as 7,000 mg/L.²⁴ Results indicated that lower doses of nitrate (e.g. 40 mg/L¹⁵) could decrease liver enzymes, whereas higher doses (e.g. 5,000 mg/L²⁰ and 7,000 mg/L²⁴) were harmful. To our knowledge, no long-term study has addressed the dose-dependent effects of sodium nitrate on serum liver function tests (LFTs). According to European Food Safety Authority (EFSA), nitrate is naturally found in various food, particularly in green leafy vegetables and beetroot, and also has a widespread use as a food additive.^26,27 In recent years, nitrate has attracted considerable attention as a potential health-promoting dietary compound.²⁷ To better understand dose-dependent safety profile and association between sodium nitrate and LFTs, this study was conducted to determine the long-term (6 mo) dose-dependent effect of sodium nitrate (50, 100, 150, 250, and 500 mg/L) on serum ALT, AST, and ALP in both female and male rats.

Materials & methods

Animals

Female and male Wistar rats (n = 120, 8-wk-old) were housed under standard laboratory conditions (temperature 21 ± 2 °C and dark/light cycles of 12 h) with free access to water and food (pellet diet from Pars Animal Feed Company, Tehran, Iran). According to the principle of the 3Rs (Replacement, Reduction, and Refinement²⁸), to reduce the number of rats used in this study, serum samples were obtained from our previous study involving nitrate's effects on adiposity in normal rats.²⁹ At the end of the mentioned study, rats were anesthetized with intraperitoneal injections of ketamine (50 mg/kg) and xylazine (10 mg/kg), before blood collection. Subsequently, rats were sacrificed humanely under anesthesia prior to tissue isolation.

Sample size calculation

Our rationale for selecting this number (n = 10/sex/dose) was based on prior studies examining nitrate's effects on body weight²⁹ and lipid profiles³⁰ in normal rats. Additionally, a pilot study evaluating the effects of nitrate (50 mg/L) on ALT observed standard deviations of 8.6 and 6.8 for the control and nitrate-supplemented groups, respectively. Since the effect size was considered to be 10%, we set a two-sided α of 0.05 and a power of 80%. The sample size in each group was calculated to be 10 according to the following formula:³¹

;

Where and are means and variances of the two groups, respectively.

The number of rats used in this study (10 rats per sex per group) is also in line with Organization for Economic Co-operation and Development (OECD) guidelines, recommending obtaining blood samples from at least 10 male and 10 female rats per group to investigate major toxic effects in the liver.³² However, OECD guidelines recommend 20 animals per sex per dose to enable comprehensive biological and statistical assessments.³²

Timeline of the study

The experimental study design and timeline are depicted in Fig. 1. A total of 120 rats (60 females and 60 males) were randomly divided into 6 subgroups (n = 10/subgroup for each sex). In both female and male rats, the control group received regular tap water, while the five treatment groups received tap water containing sodium nitrate at concentrations of 50, 100, 150, 250, and 500 mg/L, respectively, for a duration of 6 mo. Fasting serum levels of ALT, AST, ALP, and NO metabolite (NOx) were measured at month 0 (baseline) and month 6 of the study.

Fig. 1.

Study timeline. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase, NOx, nitric oxide metabolites.

Measurement of liver enzymes

Serum levels of ALT, AST, and ALP were measured at baseline (months 0) and after 6 mo of treatment. After an overnight fasting (8 pm to 8 am), blood samples were collected from the tail tips of anesthetized rats (intraperitoneal injection of ketamine and xylazine at dose 50/10 mg/kg) and centrifuged for 10 min at 5000 g; the sera used for analysis. Liver enzymes were measured using an autoanalyzer (Selectra-E, Netherlands) with commercial kits (Pars Azmoon Co., Iran), with sensitivities of 4, 2, and 3 international units (IU) per liter (L), for ALT, AST, and ALP respectively. The intra- and inter-assay coefficients of variation (CV) were 2.1% and 2.4% for ALT, 2.9% and 3.0% for AST, and 2.6% and 2.8% for ALP, respectively.

Measurement of serum NOx

Serum concentrations of NO metabolites (nitrate + nitrite = NOx) were measured using a modified Griess method.³³ Briefly, NaOH (3.72 M) and zinc sulfate (15 mg/mL) were added to 300 μL of serum samples to prevent turbidity during the Griess reaction and to deproteinize the samples, respectively. The samples were centrifuged at 10,000 g for 10 min, and the supernatants were used for analysis. NOx concentrations were determined by adding 100 μL vanadium trichloride to reduce nitrate to nitrite (8 mg/mL in 1 M HCl), 50 μL N-(1-naphthyl) ethylenediamine (0.1% in ddH2O), and 50 μL sulfanilamide (2% in 5% HCl) to 100 μL serum. NOx concentrations were determined using a standard calibration curve ranging from 0 to 100 μM for sodium nitrate. The concentrations of NOx in the serum are expressed as μmol/L. The intra- and inter-assay CVs were 4.7 and 5.9%.

Statistical analysis

Data are reported as mean ± SEM. Statistical analysis was performed using Graph Pad Prism software, version 8. To investigate the association between different doses of sodium nitrate and serum levels of ALT, AST, and ALP, a non-linear regression analysis (dose–response model) was conducted to provide insights into the potential risks and benefits associated with varying doses of sodium nitrate administration on liver enzyme levels and to identify no observed adverse effect level (NOAEL). A two-dimensional plot with different doses on the X-axis and the corresponding biological responses on the Y-axis was constructed. We plotted the percent change in ALT, AST, and ALP at month 6 on the Y-axis, using their corresponding baseline values as reference levels, and the X-axis represented increasing sodium nitrate doses (50, 100, 150, 250, and 500 mg/L). In addition, the correlation between ALT, AST, and ALP levels and serum NOx concentration was determined using Spearman correlation analysis. Two-sided P-values<0.05 were considered to be statistically significant.

Results

J-shaped association between sodium nitrate doses and serum liver enzymes

The dose–response relationship between sodium nitrate and LFTs demonstrated a hormetic (J-shaped) association in both female and male rats (Fig. 2). Untreated female rats exhibited increases in serum ALT, AST, and ALP levels at month 6 compared to baseline (month 0), with respective increases of 11.5%, 9.5%, and 15.7% as indicated by horizontal dotted lines in Fig. 2A–C. Similarly, untreated male rats showed respective increases of 7.5%, 9.6%, and 16.8% (Fig. 2D–F). Sixth months of sodium nitrate administration at the highest dose (500 mg/L) resulted in significant increases in serum ALT, AST, and ALP levels, with female rats showing increases of 27%, 26%, and 30%, respectively, and male rats showing corresponding increases of 21%, 26%, and 30%.

Fig. 2.

The dose-dependent hormetic effect sodium nitrate on serum liver function tests in female (A, B, and C) and male (D, E, and F) rats. Absolute baseline values for ALT (A and D) are 33 ± 0.7 and 33 ± 0.8 U/L, for AST (B and E) are 86 ± 2 and 88 ± 2 U/L, and for ALP (C and F) are 250 ± 10 and 257 ± 8 U/L, in female and male rats, respectively. In addition, the % of the change in the Y axis showed a change of LFTs during 6 mo of treatment (month 6 vs. month 0). NOAEL, no observed adverse effect level; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

The hormetic zones for sodium nitrate doses, indicating beneficial effects, and toxic zones, indicating detrimental effects, were identified for ALT, AST, and ALP in both sexes. For ALT, the hormetic zone was 10–230 mg/L in females and 5–190 mg/L in males, with toxic zones above 230 mg/L and 190 mg/L, respectively (Fig. 2A and D). For AST, the hormetic zone was 20–180 mg/L in females and 7–180 mg/L in males, with toxic zones above 180 mg/L in both sexes (Fig. 2B and E). For ALP, the hormetic zone was 8–270 mg/L in females and 7–265 mg/L in males, with toxic zones above 270 mg/L and 265 mg/L, respectively (Fig. 2C and F).

These findings underscore a dose-dependent dual effect of sodium nitrate on liver enzyme activity, with beneficial effects observed at low-to-moderate doses and toxic effects emerging at higher doses.

Correlation between serum NOx and ALT, AST, and ALP

The relationship between serum NOx concentrations and liver enzymes exhibited a dose-dependent pattern. At sodium nitrate doses of 100 mg/L and 150 mg/L, serum NOx levels were negatively correlated with ALT (r = −0.498, P = 0.001, n = 40; Fig. 3A) and AST (r = −0.320, P = 0.044, n = 40; Fig. 3B), suggesting protective effects at these doses. In contrast, at higher doses of 250 mg/L and 500 mg/L, serum NOx showed a positive correlation with ALT (r = 0.500, P = 0.001, n = 40) and AST (r = 0.300, P = 0.059, n = 40), indicating a shift toward potentially harmful effects. Serum NOx was not correlated with serum ALP levels in either female or male rats at any dose. These findings highlight the complex, biphasic nature of sodium nitrate's effects on liver function and the role of NOx in mediating these effects.

Fig. 3.

Correlation between serum ALT (A), AST (B), and ALP (C) and nitric oxide metabolite concentration (NOx) in nitrate-treated groups received tap water containing sodium nitrate for 6 mo. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

Discussion

The present study showed a J-shaped dose-dependent effect of long-term sodium nitrate administration on serum ALT, AST, and ALP in female and male rats. A significant negative correlation was observed between serum NOx and ALT and AST levels at moderate doses (100 and 150 mg/dL), transitioning to a positive correlation at higher doses (250 and 500 mg/dL). This biphasic response supports a hormetic association between sodium nitrate and liver enzymes, where specific doses yield protective effects, while higher doses exhibit toxicity. The baseline liver enzyme levels in this study align well with previously reported normal ranges for ALT, AST, and ALP in rats: approximately 50–190 U/L for ALT, 60–150 U/L for AST, and 80–400 U/L for ALP.^{15,20,22–25,32}

Previous studies on the effects of nitrate on liver enzymes, summarized in Table 1, demonstrate variability in findings—ranging from decreases¹⁵ to increases²⁴ or no significant effect.²⁵ In these studies, doses of nitrate ranged between 40 mg/L (~7 mg/kg)¹⁵ to 7,000 mg/L (~1,200 mg/kg)²⁴) as a single,^15,21,23,24 two,²⁵ or three^20,22 doses administrated to male rats for 10–60 days^20–25,35 and male mice for 210 days.¹⁵ It should be noted that most of these studies focused exclusively on male animals, employed shorter durations, and utilized a limited range of nitrate doses. Our findings for the first time indicate that nitrate has a hormetic effect on serum liver enzymes, which explains these inconsistent findings and provides a framework for understanding nitrate's dual effects.

Table 1.

Findings of studies showing the effect of nitrate on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in healthy rodents.

Study	Year	Animal	Sex	Age a (weeks)	Intervention	Administration route	Dose		Duration (days)	Liver enzymes
							mg/L	mg/kg		ALT	AST	ALP
Ogur, et al.25	2005	Sprague–Dawley rats	Male	12	Sodium nitrate	Tap water	200	-	60	↔	↔	↔
							400	-	60	↔	↔	↑(73%)
Azeez, et al.24	2011	Wistar rats	Male	12–16	Potassium nitrate	Food	-	1,200	42	↑(23%)	↑(39%)	NR
Bouaziz-Ketata, et al.23	2014	Wistar rats	Male	Adult b	Sodium nitrate	Intragastric tube	400	-	50	↑(29%)	↑(20%)	↑(30%)
Wang,et al.15	2018	C57BL/6 mice	Male	8	Sodium nitrate	Tap water	40	-	210	↓(23%)	↓(22%)	NR
González Delgado, et al.22	2018	Wistar rats	Male	12–16	Sodium nitrate	Intragastric tube	-	19	10	↔	↔	NR
							-	66	10	↑(23%)	↔	NR
							-	150	10	↔	↑(43%)	NR
Samia, et al.20	2023	Wistar rats	Male	6–8	Calcium nitrate	Oral gavage	-	200	30	↔	↔	↔
							-	400	30	↔	↔	↔
							-	800	30	↑(16%)	↑(17.5%)	↑(17%)
Present study	2024	Wistar rats	Female and male	8	Sodium nitrate	Tap water	50	-	180	Sub-hormetic dose
							100	-	180	Hormetic doses
							150		180
							250	-	180	Toxic doses
							500	-	180

Abbreviation: NR, Not reported.

^aAt the start of the nitrate administration.

^bThe exact age of the animals was not reported in this study. Usually, rats >10 wk are considered adults.³⁴

A hormetic association between sodium nitrate and liver enzymes in female and male rats, found in our study, illustrates that the dose–response association is non-linear and can shift from beneficial to adverse effects with increasing dose. This association can be categorized into three zones. In the sub-hormetic (<10 mg/L for all three enzymes), nitrate had no significant effects. The hormetic (100 mg/L for ALT and AST and 150 mg/L for ALP) showed optimal benefits, while the toxic zone (>200 mg/L for ALT and AST, >250 mg/L for ALP) demonstrated detrimental effects. Our results align with previous studies reporting toxic effects at doses exceeding 200 mg/L (~30 mg/kg) (Table 1). For instance, nitrate doses of 400 mg/L,^22,23,25 1,000 mg/L,²² 5,000 mg/L,²⁰ and 7,000 mg/L²⁴ corresponding to ~66, 150, 800, 1,200 mg/kg, respectively, increased AST by 17%,²¹ 20%,²³ 39%,²⁴ and 43%²²; ALT by 23%,^22,24 29.5%,²³ and 45%²¹; and ALP by 30%,²³ and 73%.²⁵ In contrast, lower doses such as 40 mg/L administered for 210-day nitrate, reduced ALT and AST by approximately 20% in male mice,¹⁵ consistent with the hormetic zone identified here.

Hormesis—a biphasic dose–response—has been highlighted by Calabrese et al.³⁶ as a fundamental principle in toxicology, particularly concerning NO and its donors. For example, NO at hormetic zone inhibits sperm apoptosis,³⁷ but at higher doses can be toxic, with optimal effects observed at concentrations between 80 to 100 nM. In the body, nitrate is converted to nitrite and then to NO. Experimental data strongly suggests that NO derived from nitrate in the hormetic zone functions similarly to eNOS-derived NO, which produces NO between 10 to 50 nM and has a protective effect on the liver. However, nitrate at the toxic zone may behave like iNOS-derived NO,¹⁹ which produces NO at levels greater than 100 nM and has detrimental effects on liver function. Nitrate within the hormetic zone enhances liver function by reducing the activity of complex I in the mitochondrial electron transport chain through nitrosylation. This reduction decreases nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) coenzyme, subsequently lowering the production of reactive oxygen species (ROS) in the liver.^38–40 The protective effects of nitrate on liver function are further attributed to its anti-inflammatory and antioxidative properties,^41–43 as well as its ability to improve blood circulation, which aids the removal of lipid peroxides.⁴⁴ In contrast, high concentrations of NO can bind to cytochrome c oxidase, leading to increased production of ROS and peroxynitrite. These reactive compounds irreversibly inhibit mitochondrial respiration, damage mitochondrial membrane and DNA, and alter mitochondrial membrane permeability, ultimately inducing cell death.⁴⁵

A strength of this study is the long-term administration of sodium nitrate (6 mo) in both sexes, an approach that is in line with the National Institutes of Health's recommendation to ensure sex balance in animal studies.⁴⁶ In addition, we concluded that nitrate doses below 200 mg/L (equivalent to 22 mg/kg/day based on water consumption and body weight in rats) are protective and have no adverse effects. When translated to humans, this dose corresponds to ~3.5 mg/kg/day in humans, which is almost identical to current acceptable daily intake (ADI) for nitrate (i.e. 3.7 mg/kg).^47,48 Daily intake of nitrate is variable across populations; EFSA notes that when considering all sources of dietary nitrate including food additives, natural presence, and contamination, a typical range of 1.5 to 8.7 mg/kg (especially in the elderly and children) is observed that can exceed the ADI value.⁴⁹ However, results of systematic review⁴⁸ of 55 observational studies involving a total of 3,430,148 participants indicate that the median nitrate intake is ~1.5 mg/kg/day (rang: 1.28 to 2.14 mg/kg/day), a value well below the ADI threshold. In 2017, a panel of experts from the EFSA, concluded that the epidemiological evidence does not support an association between nitrate intake above the ADI value and cancer risk,⁴⁹ an issue that is also supported by results of meta-analyses.⁵⁰ While the WHO's ADI of 3.7 mg/kg/day remains in place, the multiple health benefits associated with nitrate consumption has led to proposing reconsidering the nitrate ADI value, particularly concerning long-term effects.^27,49 As a limitation, we did not investigate the potential molecular mechanisms underlying the beneficial and detrimental effects of nitrate on liver enzymes. Beneficial effects of low amount of nitrate-derived NO include activation of the ERK,⁵¹ PI3K/Akt,⁵² and soluble guanylate cyclase (sGC)-cGMP⁵³ signaling pathways that reduces oxidative stress, inflammation, and apoptosis in the liver. In contrast, high amount NO increases reactive nitrogen species such as peroxynitrite (ONOO⁻), p53 expression, and inflammation that cause oxidative stress, mitochondrial dysfunction, and apoptosis that eventually lead to liver injury^19,54,55

Conclusions

A J-shaped dose-dependent effect of sodium nitrate on serum liver enzymes was identified in both female and male rats, demonstrating distinct ineffective, therapeutic, and toxic dose ranges. In both healthy female and male rats, for ALT and AST, the optimal protective dose was 100 mg/L, with doses lower than 10 mg/L being ineffective and doses exceeding 200 mg/L being associated with toxicity. For ALP, the best protective effect was observed at 150 mg/L, while doses below 10 mg/L were ineffective and those above 250 mg/L exhibited toxic effects. These findings provide a mechanistic understanding of the dual beneficial and detrimental effects of sodium nitrate in healthy rats and clarify the varying outcomes reported in previous studies that employed single-dose administration. Additionally, further studies are needed to evaluate the potential therapeutic applications of sodium nitrate in pathological conditions to better understand its role and implications in disease management.

Author contributions

R.Z.: Investigation, Methodology, Performed the experiments, Writing - Original Draft, S.J.: Investigation, Performed the experiments, Interpreted the results, Writing- Original draft, K.K.: Conceptualization, Interpreted the results, Writing - Original Draft, A.GH.: Conceptualization, Methodology, Project administration, Supervision, Interpreted the results, Writing- Original draft. All authors reviewed the manuscript.

Funding

This study was supported by a grant (Grant No. 43011048–3) from Shahid Beheshti University of Medical Sciences. KK, supported in part by the National Institutes of Health, USA; grant numbers R01GM123508 and 2U54MD017979-01A1.

Conflicts of interest

The authors declare that they have no competing interests to disclose.