Bioassays to Assess the Safety of Potassium and Sodium Nitrates and Nitrites

Abstract

(1) Background: Advances in food processing practices and health care are some of the most significant advances in modern daily life. The goal of this study is to evaluate the safety of potassium and sodium nitrates and nitrites when they are used as fertilizers in agriculture and food additives, as well as the known conversion of nitrate to nitrite in humans. (2) Methods: Various bioassays were conducted to investigate the effects of nitrates and nitrites in the Drosophila melanogaster genetic tester system. These assays focused on the modulation of degenerative processes at the molecular, cellular, individual, and population levels. Additionally, we assessed the chemopreventive potential and the ability to induce DNA strand breaks in HL-60 tumour cells. (3) Results: All nitrate and nitrite concentrations tested were shown to not be toxic or genotoxic in Drosophila since none of the compounds reached the LD₅₀ and significant genetic mutation. A positive or null protective capacity against a toxic agent was found for nitrates, not for nitrites, showing that sodium nitrite has a synergistic effect when combined with the oxidant toxin hydrogen peroxide; and a nutraceutical potential in the lifespan only for sodium nitrate to improve the quality of life in 5 days at ADI concentration. The in vitro results in human leukemia cells showed a chemopreventive potential only for potassium nitrate and sodium nitrite due to reducing the viability of HL-60 cells growth to 18% and 29%, respectively, compared to the controls at ADI (acceptable daily intake) concentrations. However, neither of these showed DNA damage or methylation modifications. (4) Conclusions: The tested compounds were shown to be safe to use during in vivo and in vitro tests when used at the extrapolated ADI concentrations.

1. Introduction

According to Stein and Klotz [1], nitrates and nitrites are ionic compounds found in nature as part of the nitrogen cycle. The main sources of nitrates and nitrites in modern diets include plants such as vegetables and fruits, including zucchini, spinach, carrots, potatoes, lettuce, beet greens, fennel, cabbage, parsley, celery, cucumbers, radishes, and leeks, as well as processed and cured meats such as bacon, mortadella, cured meat, ham, salami, sausages and preserves, a variety of fish and poultry, and water (especially if there is runoff or nitrate or nitrite contamination from agricultural sources) [2,3,4,5,6,7].

Nitrate is a very common fertilizer in agriculture that can lead to biological problems in the environment [8]; drinking water may contain nitrates and/or nitrites from fertilizer residues in agricultural communities [5]. A considerable amount of water is used during the preparation and cooking of food such as fruits, vegetables, stews, soups, desserts, coffee, tea, and frozen foods. Nitrate is mainly derived from manure as ammonium, sodium, potassium, and calcium salts [9]. Nitrate is primarily absorbed via drinking water, vegetables, and other foodstuffs; it is not produced naturally in the body in significant amounts [10]. Due to the harmful effect that these compounds have on the body in high doses, their ADI was established by the Joint Committee of the FAO/WHO with a value between 0 and 3.7 mg/kg × bw/day [11]. Its toxicity comes from its conversion into nitrites and the possible endogenous formation into N-nitroso compounds [12,13].

On the other hand, nitrites are used in food processing as additives; in addition to providing the right color to certain meat products, they have other effects on food: they delay the oxidation process of lipids, with the consequent reduction of the characteristic rancid smell, produce a firmer texture, and provide foods with an important antimicrobial effect (especially against Clostridium botulinum and its toxins) [14,15]. Other uses include the manufacture of explosives and the maintenance of industrial boilers [16]. The ADI for nitrites recommended by the Joint Committee of the FAO/WHO is 0–0.06 mg/kg × bw/day [17]. The use of nitrite as an additive in baby food for children under three months is not permitted [8]. Moreover, it is known that nitrate is converted to nitrite compounds in humans, the final product being 10 times as toxic as nitrate in humans [18]. In general, plants are the primary sources of nitrates, while processed and cured meats are the primary sources of nitrites [6].

In this article, the biological effects that different concentrations of potassium and sodium nitrate and nitrite, as food additives, show on complete animal eukarya organisms and unicellular systems are explored to add a new scope to determine the modulator role of this important group of compounds in nutraceuticals.

Potassium nitrite (E-249) can be produced by reducing potassium nitrate, similar to the process used for sodium nitrite. Although it is used only in small quantities, potassium nitrite serves important functions, such as acting as an oxidizing agent and as a corrosion inhibitor. Potassium nitrite is more soluble than sodium nitrite and does not form sparingly soluble bicarbonate in carbondioxide-rich solutions [19].

Sodium nitrite (E-250) is the most significant salt of nitrous acid in industrial applications. While nitrites do not naturally occur in mineral form, they are produced as metabolic byproducts by microorganisms that oxidize organic nitrogen-containing substances. As a result, small amounts of nitrite can be found in soil and groundwater. Until the early 20th century, sodium nitrite was primarily produced by reducing sodium nitrate. However, large-scale production of sodium nitrite now relies on the reaction of nitrogen oxides with sodium carbonate or sodium hydroxide solutions. Chemical and pharma industries are the principal consumers of sodium nitrite: dyes, pesticides like pyramin, caffeine, lubricants, and other applications are manufactured using large amounts of this compound. Other applications of sodium nitrite are in metallurgy and corrosion prevention, and it is used in lubricants for glass-forming equipment (as sodium nitrate). Sodium nitrite is recommended as an additive: in curing salts used in the food industry, it is still the most reliable agent for protecting against botulism, a dangerous bacterial contamination of meats. High nitrite concentrations can, however, lead to the formation of nitrate and nitrite carcinogenic nitrosamines [19].

Sodium nitrate (E-251) is the only abundantly occurring mineral nitrate. Fifty percent of sodium nitrate produced worldwide is used as a fertilizer for crops, and the leftover amounts of sodium nitrate are used in the explosives industry, pyrotechnics, glass and enamel industry, metallurgy, solar technology, to promote combustion, production of dyes, pharmaceuticals, charcoal briquettes, and other nitrates [19]. Directly related to human health, uses of sodium nitrate are pleiotropic: Clostridium botulinum proliferation, in cyanide poisoning intoxications, as antihypertensive, and cardiovascular diseases [19,20,21,22,23,24].

Potassium nitrate (E-252) occurs as efflorescence on soils in mineral niter formula and is used only for gunpowder production. Most potassium nitrate is manufactured to be used as a non-hygroscopic fertilizer and in the production of clear liquid fertilizers and is a crucial element of multi-nutrient fertilizers. Potassium nitrate is also used in metallurgy, in the manufacture of explosives and pyrotechnics, in food preservation (like cheese processing and improving the quality of tobacco), can be used for desooting in combustion processes or as an oxidant in chemical syntheses, and has been proposed as an oxidizing component in an acid-based gas generator systems for the rapid inflation of airbags [19].

Model organisms play a crucial role in understanding the effects of potassium and sodium nitrates and nitrites. In vivo studies with fruit flies have demonstrated that a diet supplemented with these additives can have both beneficial and harmful effects on Drosophila, depending on the dosage consumed [25,26,27,28]. Additionally, it is important to consider a variety of in vitro studies that examine the impact of nitrite and nitrate consumption on different types of cancer cells for health implications [11,17,29,30,31].

Taking into account the accessible information on different nitrates and nitrites, our purpose is to assess, in a transversal way, the biological effects that the aforementioned compounds have in degenerative processes related to aging, as well as providing a new data set to the field and contributing to wider scientific progress. As a result, a comprehensive study of biological activity was conducted at individual, cellular, and molecular levels, utilizing in vivo and in vitro assays with two model systems: Drosophila melanogaster and HL-60 cell line.

2. Materials and Methods

2.1. Samples

Potassium and sodium nitrates and nitrites were selected according to their abundance and importance in agriculture and diet for human consumption. With the aim of understanding the biological effects at different purpose levels, a set of six concentrations was tested for each compound. The concentrations were determined based on the daily food intake of Drosophila melanogaster, which is 1 mg/day, and the average weight of individual D. melanogaster, also 1 mg [32]. These concentrations were calculated to be comparable to the acceptable daily (ADI) for humans [11,17], as indicated in

Table 1. Compounds information.

Compound		Concentrations *
		1	2	3	4	5	6
E-249	Potassium Nitrite (Cat. 12654)	0.0000001	0.000001	0.00001	0.0001	0.001	0.01
E-250	Sodium Nitrite (Cat. 67398)	0.0000001	0.000001	0.00001	0.0001	0.001	0.01
E-251	Sodium Nitrate (Cat. 15736)	0.0000185	0.000185	0.00185	0.0185	0.185	1.85
E-252	Potassium Nitrate (Cat. 542020)	0.0000185	0.000185	0.00185	0.0185	0.185	1.85

* Concentrations numbered 1 to 6 represent the value, in mg/mL, of the different dilutions tested; the equivalent amount to ADI in humans is number 3. All compounds were obtained from Sigma (Darmstadt, Germany).

.

2.2. In Vivo Assays

The holometabolous eukaryote Drosophila melanogaster is used as the in vivo tester system. The characteristics of its life cycle are well known as well as the similarities with human diseases and the reliability of test results [33,34,35,36,37,38]. Two conventional Drosophila genetic tester strains were used (mwh/mwh and flr³/In (3LR) TM3, rip^p sep bx^34e e^sBd^S) [39,40,41].

2.2.1. Toxicity

The safety of Drosophila flies is evaluated by measuring their survival percentage against the concurrent negative control when treated with different concentrations of nitrates and nitrites (see Table 1).

The number of emerged adults in each treatment was analyzed at various concentrations of nitrates and nitrites (see Table 1). Each tube contains Drosophila Instant Medium (DIM) (Formula 4–24, Carolina Biological Supply, Burlington, NC, USA) and 4 mL dilutions of the different compounds. Additionally, negative controls were prepared using DIM mixed with distilled water.

The toxicity assays were conducted over three independent experiments. The statistical significance of the results in relation to the concurrent controls was analyzed using the formula [(nº of individuals hatched in each treatment/nº of individuals hatched in the concurrent control) ×100]. Statistical analysis was performed using the non-parametric Chi-square test [42].

2.2.2. Antitoxicity

In the antitoxicity test, the protective ability of these compounds on the flies when treated with a toxin is evaluated. The survival percentage of individuals is evaluated against their concurrent positive controls when Drosophila is treated with different concentrations of nitrates and nitrites (see Table 1) combined with 0.12 M H₂O₂ (Sigma Chemical CO., St. Louis, MO, USA, H1009) as a toxicant [43].

The statistical significance of three independent experiments was evaluated by the non-parametric Chi-square test [42].

2.2.3. Genotoxicity

Following the method described by Graf et al. [44], in this assay, we evaluated the genomic safety at the chromosomal level by measuring the SMART (somatic mutation and recombination wing spot test) of Drosophila treated at the larval stage with different ADI concentrations of nitrates and nitrites (see Table 1).

Dilutions were prepared as in the toxicity test, and a positive control was included, consisting of DIM, water, and 0.12 M H₂O₂ as a genotoxicant to check the reproducibility of the assay. Transheterozygous wings were subsequently mounted for scoring mutations at 400× magnification.

The total mutations were categorized based on their size and type as they correspond to different mutational events [44].

A total of 234 wings were mounted and analyzed. Additionally, a double-decision statistical test was applied [45,46]. To address the inconclusive and positive results, the non-parametric Mann–Whitney U-test (α = β = 0.05) was used.

2.2.4. Antigenotoxicity

The protective potential at the chromosomal level is evaluated in treated Drosophila larvae by the SMART with the ADI concentration of nitrates and nitrites (see Table 1) combined with H₂O₂.

Dilutions were prepared as in the antitoxicity assays as described by Graf et al. [47]. Once adult flies emerged, a total of 186 wings were mounted and analyzed using the same genotoxicity protocol. The inhibition percentages of mutagenic activity (IP) were calculated according to the algorithm outlined by Abraham [48]:

IP = [(genotoxin—combined treatment)/genotoxin] × 100

2.2.5. Longevity

The evaluation of effects produced by nitrates and nitrites at different concentrations on the lifespan and quality of life of Drosophila populations when they are chronically fed were analyzed in this assay.

The methodology for longevity trials was in detail formerly described by our research group [49].

The healthspan, or quality of life, of treated flies was evaluated by analyzing the upper 25% of the survival curve, which is characterized by a low and relatively constant age-specific mortality rate [50].

To analyze the survival curves and identify differences among them, we utilised Kaplan–Meier methodology using SPSS Statistics 17.0 software (SPSS, Inc., Chicago, IL, USA) along with the log-rank (Mantel–Cox) test.

2.3. In Vitro Assays

Cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA, R5886) supplemented with additional components at standard conditions (37 °C and 5% CO₂) [51] using a ShellLab (Cornelius, OR, USA) CO₂ incubator.

2.3.1. Cytotoxicity

In this assay, the ability of nitrates and nitrites to inhibit the tumour cell growth was analyzed with 72 h treatments at different concentrations of nitrates and nitrites (see Table 1).

To assess cell viability, the trypan blue exclusion test (Sigma-Aldrich, St. Louis, MO, USA, T8154) was employed. The results were represented graphically as a percentage of survival from three independent experiments compared to their concurrent control.

2.3.2. Internucleosomal DNA Fragmentation

DNA damage caused by the nitrates and nitrites was assessed at the internucleosomal level by measuring proapoptotic DNA fragmentation in this assay.

HL-60 cells treated with various concentrations of nitrates and nitrites (see Table 1) were centrifuged, and DNA was extracted following the protocol outlined by Merinas-Amo et al. [52].

The extracted DNA was quantified using a spectrophotometer (Nanodrop ND-1000) and, then, subjected to agarose gel (2%) electrophoresis where it was visualized under UV light.

2.3.3. Comet Assay

DNA damage in single tumour cells caused by comet formation was evaluated following treatment with the ADI concentration of nitrates and nitrites (see Table 1). The assessment began with DNA extraction, utilizing the same protocol as in the DNA fragmentation assay.

As outlined in the protocol by Mateo-Fernández et al. [53], the process involved several key steps: washing, freezing, lysis, alkaline electrophoresis, neutralization, and drying. The single cells were then visualized using a Leica DM 2500 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Parameters were calculated using the OpenComet plugging in ImageJ (NIH).

Statistical analysis of the tail moment was conducted using ANOVA and a post hoc Tukey test, with results processed using SPSS Statistic 17.0 software.

2.3.4. Methylation Status

In this assay, we evaluated the ability to modulate epigenome by studying the methylation status pattern of genome-wide sequences, specifically, Alu, LINE, and Satellite repetitive elements, in tumor cells treated with the ADI concentration of nitrates and nitrites (see Table 1).

After extraction of treated DNA using the same method outlined in the fragmentation protocol, we performed a bisulphite modification of the DNA. This was followed by quantitative methylation-specific PCR (qMSP) and an analysis method as described by Merinas-Amo et al. [54].

We were able to evaluate a wide range of human genomic DNA across repetitive sequences. This is possible because Alu and LINE sequences are interspersed throughout the genome, while satellite sequences are concentrated in the centromeric regions [55,56,57,58]. The Alu M1, LINE-1 and Sat-α sequences were obtained from Isogen Life Science B.V. (Utrecht, The Netherlands)(refer to

Table 2. Information of repetitive sequences used for the evaluation of methylation status modulation.

Reaction ID	Gene Bank Access	Amplicon		Sequence 5′ to 3′ First Forward	Sequence 3′ to 5′ First Reverse	GC-Content
		Start	End			Forward	Reverse
				GGTTAGGTA	ATTAACTAAA
Alu C4	Sequence	1	98	TAGTGGTTTA	CTAATCTTAA	25	27.3
	Consensus			TATTTGTAAT	ACTCCTAACC
				TTTAGTA	TCA
				ATTATGTTAG	CAATCGACC
Alu M1	Y07755	5059	5164	TTAGGATGG	GAACGCGA	27.6	58.8
				TTTCGATTTT
				GGACGTATT	AATCTCGCGA
LINE-1	X52235	251	331	TGGAAAATC	TACGCCGTT	47.6	52.6
				GGG
				TGATGGAGT	AATTCTAAAA
				ATTTTTAAAA	ATATTCCTCT	23.5	21.2
Sat-α	M38468	139	260	TATACGTTTT	TCAATTACGT
				GTAGT	AAA

Source: Weisenberger et al. [59].

for further information [59]).

In this assay, we evaluated the ability to modulate the epigenome by analyzing the relative expressions obtained from three replicas of each sample. These results were normalized using the Alu C4 housekeeping sequence, following the comparative C_T method outlined by Nikolaidis et al. [60] and Liloglou et al. [61]. To assess statistical differences between the control group and each treated group, we employed one-way ANOVA along with the post hoc Tukey test.

3. Results

3.1. Toxicity

The results of the toxicity tests for each of the substances studied are represented in Figure 1. In general, none of the additives, except the highest concentration of sodium nitrite tested, reached the considered toxic lethal dose 50 (LD₅₀). In addition to the validity of the test, this corroborates that the ADI concentration (numbered as 3) established by JECFA for each compound studied is a secure dose [11,17,62].

The toxicity effects of each compound in the model organism indicated:

• Potassium nitrite: It presents a dose-dependent effect on toxicity, reaching slightly harmful results for Drosophila at the three highest concentrations studied. The viability range was modified between 80% and 72% compared to its control.
• Potassium nitrate: It showed a significant decrease in the survival of individuals treated at concentrations 4 and 5, seeing their viability decrease to 84% and 75%, respectively, compared to the control.
• Sodium nitrite: It exhibited a dose-dependent toxic effect, with a significant decrease in survival at the three highest concentrations studied, the highest concentration being the only one that shows a significantly toxic effect for Drosophila with respect to the control (under the LD₅₀). The viability range decreased between 84% and 39% with respect to its control.
• Sodium nitrate: The results show a dose-dependent toxic effect, with the two highest concentrations studied showing a significant decrease in survival of individuals, although none of them reached LD₅₀.The viability percentage decreased to 75% and 84%, respectively, compared to the control.

3.2. Antitoxicity

The antitoxicity results (Figure 2) showed that not all the additives selected in the study have the capacity to protect Drosophila from oxidative stress generated by hydrogen peroxide. The observed discrepancy between the results of toxicity and antitoxicity tests on the food additives studied may arise from the fact that each substance can exhibit either antioxidant or pro-oxidant activities. This competition occurs against the effects of hydrogen peroxide when the two are combined [63].

The potential protective, null, or harmful effect of the selected compounds when combined with a toxic oxidizing agent is detailed below.

• Potassium nitrite: It did not show significant capacity for the protection of this additive against a stressor when combined in the model organism D. melanogaster.
• Potassium nitrate: The two minimum concentrations tested showed a significant protective effect with an increase in the survival of individuals of 19% and 17%, respectively, with respect to the positive control when combined treatments were carried out with a genotoxic agent. However, the highest concentration studied showed a significant negative antitoxic effect on Drosophila, with a decrease in the survival of individuals of 12% compared to the positive control.
• Sodium nitrite: It exerted a significant antitoxic effect for the two lowest concentrations studied in D. melanogaster, with an increase in viability of 22% and 12%, respectively compared to the positive control. The rest of the concentrations tested did not show significant effects on the survival of the flies.
• Sodium nitrate: The results exhibited a significant antitoxic effect at the three lowest concentrations studied in Drosophila, with an increase in the percentage of viability between 8% and 15% compared to the positive control and a significant negative effect on the survival of individuals in combined treatments at a high concentration of this additive, with a decrease in viability of 15% compared to the positive control.

3.3. Genotoxicity

Results of the SMART genotoxicity test for nitrate and nitrite treatments at the ADI concentration are indicated in

Table 3. Genotoxicity in Drosophila melanogaster wing spot test treated at ADI concentrations.

	Clones Per Wings (Number of Spots) (1)
Compound	Wings Number	Small Single Spots (1–2 Cells) m = 2	Large Single Spots (>2 Cells) m = 5	Twin Spots m = 5	Total Spots m = 2	Mann–Whitney U-Test (2)
H2O	38	0.105 (4)	0.053 (2)	0	0.158 (6)
H2O2	40	0.200 (8)	0.200 (8)	0	0.400 (16) +
Potassium Nitrite	40	0.150 (6)	0.050 (2)	0	0.200 (8) i	Δ
Potassium Nitrate	36	0.166 (6)	0.000 (0)	0	0.166 (6) i	Δ
Sodium Nitrite	40	0.225 (9)	0.100 (4)	0	0.325 (13) i	Δ
Sodium Nitrate	40	0.175 (7)	0.025 (1)	0	0.200 (8) i	Δ

⁽¹⁾ Statistical diagnosis was conducted according to the methods of Frei and Würgler [45]. Results were characterized as positive (+) or inconclusive (i) compared to the negative control. The multiplication factor is denoted as “m”. The Kastenbaum–Bowman test was performed without the Bonferroni correction, with probability levels set at α = β = 0.05. ⁽²⁾ The Mann–Whitney U-test was utilized when necessary to clarify inconclusive results. The Delta symbol (∆) indicates that there were no significant differences compared to the negative control.

.

The total frequency of the number of clones shown for the negative and positive controls was 0.158 and 0.400, respectively. These values are within the range established in similar previous studies when using the same genetic background [64,65]. Furthermore, the selected concentration of H₂O₂ (0.12 M) has been shown to induce mitotic recombinations and somatic mutations in Drosophila melanogaster [64].

The mutation frequency range for the set of additives studied, at their ADI concentration, is between the values of 0.166 and 0.325 clones/wing related to the negative control. After analyzing the results with the Mann–Whitney U-test, none of the results were statistically significant with respect to the control; therefore, there are no significant genotoxic effects of these additives on the Drosophila melanogaster organism.

3.4. Antigenotoxicity

Table 4. Antigenotoxicity in Drosophila melanogaster wing spot test treated at ADI concentrations.

	Clones Per Wings (Number of Spots) (1)
Compound	Wings Number	Small Single Spots (1–2 Cells) m = 2	Large Single Spots (>2 cells) m = 5	Twin Spots m = 5	Total Spots m = 2 a b		Mann–Whitney U-Test (2) a b		IP (3)
H2O	38	0.105 (4)	0.053 (2)	0	0.158 (6)
H2O2	40	0.200 (8)	0.200 (8)	0	0.400 (16)	+
Potassium Nitrite	39	0.282 (11)	0.077 (3)	0.026 (1)	0.385 (15) i	+	Δ		3.75
Potassium Nitrate	31	0.322 (10)	0.033 (1)	0	0.355 (11) i	i	Δ	Δ	11.25
Sodium Nitrite	12	0.416 (5)	0	0	0.416 (5) i	+	Δ		−4.00
Sodium Nitrate	26	0.154 (5)	0.038 (1)	0	0.192 (5) i	i	Δ	Δ	52.00

⁽¹⁾ Statistical diagnosis was conducted following the methods of Frei and Würgler [45]. Results were categorized as positive (+) or inconclusive (i) compared to the negative (H₂O) or positive control (H₂O₂). The multiplication factor is denoted as “m”. The Kastenbaum–Bowman test was applied without Bonferroni correction, with probability levels set at α = β = 0.05. ⁽²⁾ The Mann–Whitney U-test was utilized when appropriate to clarify inconclusive results, with the designations of a (negative) and b (positive). The Delta symbol (∆) indicates no significant differences when compared to the concurrent control, noted as a (negative) and b (positive). ⁽³⁾ The percentage of inhibition for the combined treatment was calculated based on the total spots per wing as described by Abraham [48].

shows the results of the SMART antigenotoxicity test for treatments with different food additives under study, at their respective established ADI concentrations (see Table 1), using the model organism Drosophila melanogaster when it is treated in combination with hydrogen peroxide as a genotoxin and with the different additives.

The total frequency of clones detected for the negative and positive controls was 0.158 and 0.400, respectively. These values are within the range established in previous studies, demonstrating that the concentration of H₂O₂ used (0.12 M) is genotoxic and is capable of inducing mitotic recombinations and somatic mutations in Drosophila melanogaster [64].

The mutation frequency range for the set of additives studied, at their ADI concentrations, is between the values of 0.192 and 0.416 clones/wing when combined treatments are carried out. Sodium nitrate had the greatest genomic protection capacity, with a 52% inhibition of mutagenic potential, followed by potassium nitrate (11.25%). However, potassium nitrite and sodium nitrite were not able to inhibit the genotoxic effects of hydrogen peroxide. The inhibition values are related to the previous partial statistical diagnosis carried out, comparing well with the positive control (no additive at the ADI concentration is statistically inferior to the positive control when performing the Mann–Whitney U-test) or comparing well with its negative control (in which case, the two nitrites studied are statistically mutagenic).

3.5. Longevity

Kaplan–Meier method curves, quality of life results (25% survival of the upper part of the longevity curve), and their significance are shown in Figure 3 and

Table 5. Mean and significance data of lifespan and healthspan assays.

Compound	Concentration (mg/mL)	Longevity (1) (Days)		Healthspan (1) (Days)
Potassium Nitrite	Control	51.355		26.569
	0.0000001	48.212	ns	26.933	ns
	0.000001	57.613	*	28.266	ns
	0.00001	43.736	ns	27.846	ns
	0.0001	64.084	*	21.900	*
	0.001	42.179	ns	28.421	ns
	0.01	55.109	ns	21.421	*
Potassium Nitrate	Control	51.355		26.569
	0.0000185	48.212	ns	24.625	ns
	0.000185	51.583	ns	30.077	ns
	0.00185	59.178	ns	23.900	ns
	0.0185	60.310	*	33.583	*
	0.185	69.333	*	31.875	*
	1.85	59.712	*	41.271	*
Sodium Nitrite	Control	51.355		25.250
	0.0000001	46.184	ns	25.769	ns
	0.000001	51.879	ns	27.167	ns
	0.00001	43.804	ns	25.789	ns
	0.0001	58.476	*	26.125	ns
	0.001	43.950	ns	24.000	ns
	0.01	53.113	ns	25.333	ns
Sodium Nitrate	Control	51.355		25.250
	0.0000185	47.596	ns	24.067	ns
	0.000185	46.205	ns	25.500	ns
	0.00185	56.347	ns	30.000	*
	0.0185	55.468	ns	25.400	ns
	0.185	59.677	*	30.900	*
	1.85	52.026	ns	27.900	ns

Values were calculated using the Kaplan–Meier method, and the significance of the curves was assessed with the log-rank method (Mantel–Cox). ⁽¹⁾ ns: non-significant, *: significant (p < 0.05).

. In general, the different compounds did not show a clear dose–effect relationship, which may suggest that there is a threshold value of significance in the population response rather than a gradation in response. The data provided to the research community by the present study area data set [11,17,66] showing no clear correlation with the concentration and the longevity of these compounds.

The most relevant data from the results of both trials are summarized below.

• Potassium nitrite: The longevity results indicated that concentrations 2 and 4 studied for this additive induce a significant improvement between 6 and 13 days of Drosophila lifespan extension when it is chronically fed. In contrast, at concentrations 4 and 6, the quality of life of these individuals is significantly reduced between 4 and 5 days compared to their control.
• Potassium nitrate: The highest concentrations tested (4, 5 and 6) indicated a significant improvement in the longevity and healthspan of Drosophila with respect to their controls between 8 and 16 days, and between 5 and 15 days, respectively.
• Sodium nitrite: The results only showed a significant improvement in 7 days of Drosophila life expansion, with respect to its control, when the chronic treatment was carried out at concentration 4.
• Sodium nitrate: The longevity of treated flies increased significantly by 8 days, compared to their control, for treatments with the concentration numbered as 5. Moreover, healthspan of Drosophila was also significantly improved in 5 days, compared to its control, when the treatments were carried out at concentrations numbered as 3 and 5.

3.6. Cytotoxicity

The cytotoxicity results for the treatments of the different compounds are shown in Figure 4. In general, considering the cytotoxic effect as a decrease in cell viability greater than 50%, we can observe cytotoxic effects in all cases except for potassium nitrite. However, analyzing the cell growth inhibition capacity of additives for the corresponding established ADI concentrations, it is observed that not only none of the additives were able to inhibit the growth of tumour cells, but some of them even stimulated such a growth.

The most relevant cytotoxicity results were as follows:

• Potassium nitrite: The results showed little cytotoxic potential of this additive, with a cell viability range between 117% and 82% compared to the control. The ADI concentration induced a stimulation of leukemia cell growth by 10% compared to untreated cells. Furthermore, the IC₅₀ was not reached, which would indicate that our additive had chemopreventive potential for any of the concentrations studied.
• Potassium nitrate: The results exhibited a dose-dependent effect on the inhibition of HL-60 cell growth. The percentage of cell viability for the different concentrations under study was between 105% and 49% with respect to the control. Concentration 3 (ADI concentration) showed an inhibition of cell growth of 18% compared to untreated cells, while the IC₅₀ was reached at the concentration 5 studied.
• Sodium nitrite: The viability percentage for cells treated with different concentrations of this additive was between 112% and 8% with respect to the control. The cytotoxic effect induced at the ADI concentration (concentration 3) indicated a cell survival of 71% related to the control. The IC₅₀ was reached between concentrations 4 and 5 under study.
• Sodium nitrate: The lowest concentrations studied showed stimulation of cell growth, reaching a viability percentage of 128% with respect to the control. Starting from concentration 3 (ADI concentration), whose viability exceeded the control growth by 13%, a dose-dependent decrease occurred until reaching a 100% cell death at the highest concentration studied. The IC₅₀ was reached between concentrations 4 and 5 studied.

3.7. Internucleosomal DNA Fragmentation

Results of genomic DNA electrophoresis of HL-60 cells treated with different concentrations of nitrates and nitrites are presented in Figure 5. At all tested concentrations of these additives, no proapoptotic damage, indicated by the lack of internucleosomal DNA fragmentation in human leukemia cells, was observed.

3.8. Comet Assay

Fabiani et al. [67] classified DNA microscopic visible damage into five classes according to the TM (comet tail) values.

Figure 6 indicates representative images of the comet test for the additives under study at their ADI concentration (concentration numbered as 3 in Table 1) as well as positive and negative controls. We evaluated the genetic damage caused by the additives in HL-60 cells using the method described by Almeida-Lima et al. [68] and Fabiani et al. [67]. We assigned a score from 0 to 4 to each cell, with 0 indicating no damage and 4 indicating severe damage. Subsequently, the statistical evaluation was carried out using the non-parametric Mann–Whitney U-test. All the results fell between the values of 0 and 1, being statistically non-significant, which indicates that no significant genomic damage was induced at the single- or double-strand level of the DNA of human leukemia cells treated with the different additives at their ADI concentrations.

The comet assay is a valuable technique that helps us identify morphological damage at the DNA level in individual cells [69]. According to Fairbairn and O’Neill [70], the TM measurements from the comet assay correlate with cytotoxicity results. Our findings support these correlations as the TM values correspond to the percentage of cell viability determined by the trypan blue exclusion test in HL-60 cells. At their ADI concentrations, none of the additives tested caused cell damage, with viability percentage ranging from 177 to 70%, compared to the control group. Therefore, the results of internucleosomal fragmentation align with those obtained from the comet as TM values less than 1 indicate that no DNA damage has occurred [67].

3.9. Methylation Status

The normalized relative expression of the three repetitive sequences studied (Alu M1, LINE1 M1 y Sat-α) in HL-60 cells treated for 5 h with the selected compounds at ADI concentrations are shown in Figure 7.

Analysis of the ANOVA and posthoc Tukey tests revealed significant changes in the methylation pattern of the tumor cells, as detailed below.

• Potassium nitrite: The results showed significant hypermethylation for Alu M1 and Sat- α repetitive sequences relative to the control.
• Potassium nitrate: All the repetitive sequences were significantly hypermethylated in treated cells, related to the control.
• Sodium nitrite: A significant desmethylation was induced in Alu M1 sequence, related to the control.
• Sodium nitrate: A significant increase of methylation in LINE M1 sequence was induced in the treated HL-60 tumor cells in relation to the untreated ones.
• Methylation of repetitive sequences is regarded as a mechanism of genomic protection by silencing key genes in the onset and progression of cancer [71,72], Potassium nitrite, potassium nitrate and sodium nitrate can be considered promising chemopreventive compounds as they have been shown to partially or completely inhibit the effects of tumor cells at the studied sequences and concentrations.

4. Discussion

Nitrates and nitrites are antimicrobial preservative additives that are added to foods for two purposes: (1) to control the natural spoilage of food and/or (2) to prevent or control contamination by microorganisms, including pathogens [73].

Although nitrates were widely used in the past, mainly as fertilizers in agriculture, their use today is restricted to slow curing of meat. Moreover, nitrate is converted to nitrite and nitrosamine compounds in humans. On the other hand, nitrites are used in meat for color formation, and flavor enhancement and to prevent antimicrobial activity. They are the only food additive that can effectively prevent the development of botulinum toxin, which supports their use when assessing benefits and risks in the food industry. In addition to serving as food preservatives, nitrites are also found in significant amounts in untreated fruits and vegetables. This occurs due to the absorption of these ionic compounds from nature through the nitrogen cycle [8]. However, the formation of nitrosamines, which are carcinogenic compounds produced when nitrites react with secondary amines, poses a potential risk to consumers [74,75,76,77,78,79].

Nitro-fatty acids (NO₂-FAs) are produced when polyunsaturated fatty acids (PUFAs) interact with reactive nitrogen species. These compounds are generated naturally during digestion, inflammation, and oxidative stress [80,81]. While the relationship between NO₂-FAs levels and metabolic diseases in humans is not well understood, studies in animal models have demonstrated that these compounds are involved in multiple protective mechanisms. NO₂-FAs have a protective role in metabolic diseases and contribute to several beneficial effects, including anti-atherosclerosis, lowering blood pressure, reducing inflammation, combating insulin resistance, and regulating glycolipid metabolism. They also show potential as therapeutic agents for a range of health issues, such as protecting cardiovascular health, supporting neurological function, safeguarding liver and kidney health, promoting skin protection, combating diabetes, reducing lipid levels, and exhibiting anti-cancer properties [82]. Despite these promising findings, further research is needed to clarify how to effectively utilize NO₂-FAs to enhance health benefits in humans.

Many factors can modify the effects of foods on general human health. There have not been any chronic assays on the effects of the aforementioned compounds in humans. In this manuscript, we evaluate the longevity of flies when fed with different concentrations of nitrates and nitrites over their lifecycle. The ADI concept gives a very high degree of protection to the consumers against exposure to nitrates and nitrites since it has been shown that high levels of these compounds can be harmful [83,84]; therefore, it is highly desirable to produce and consume foods with reduced amounts of nitrate.

Due to different sources of nitrate and nitrite exposure in humans, their safe and nutraceutical potential is studied both in vivo and in vitro model organisms. Below, we will analyze in detail the results obtained for each compound and discuss the effects obtained with similar studies.

(1)

Nitrites

The conversion of nitrate to nitrite in human saliva is the main source of nitrites coming from food and water [17] and is estimated to range from 5% to 36% [85,86,87,88,89].

Some authors consider that nitrites are carcinogenic, while others refute this possibility [90], and some even consider that vegetable nitrites are crucial for several physiological functions [23]. Furthermore, several studies in humans have shown that dietary supplementation with inorganic nitrate leads to a reduction in oxygen consumption at maximal exercise and to maintaining or even increasing work performance [91]. Although evidence supports both theories, it is widely accepted that excessive intake of nitrite is dangerous and has harmful effects on human health related to methemoglobinemia [92].

Acute toxicity effects of potassium and sodium nitrite included relaxation of smooth muscle, vasodilatation, and consequently, a lowering of blood pressure and methemoglobinemia. In humans, oral LD₅₀nitrite doses have been reported to be in the range of 100–200 mg/kg × bw (similar to bioassays in animals although in a wider range) [93].

Sodium nitrite showed mutagenic results in the SMART test at 72.5 mM [27] and at 50 mM of potassium or sodium nitrite [28].

Study of feeding female flies a nitrite-supplemented diet showed that verylow dosesof nitrite (0.1 and 1 µM) extend lifespan and favor healthspan in Drosophila [26].

Available in vitro studies provide strong evidence of the genotoxic activity of potassium and sodium nitrite. These studies show positive results in tests for gene mutations in bacteria, as well as in tests for the induction of structural chromosomal aberrations, gene mutations, aneuploidy, and cell transformation in mammalian cells [17]. In contrast, in vivo studies have yielded negative results in well-conducted micronucleus assays performed on mice and rats despite measurable systemic exposure following acute and subchronic administration of sodium nitrite. There are also limited negative data regarding effects at the site of contact. Overall, the panel concluded that the available information does not indicate an in vivo genotoxic potential for sodium and potassium nitrite. Consequently, this does not rule out the possibility of establishing a health-based guidance value (ADI) [17].

In tests using cultured mammalian cells without metabolic activation, sodium nitrite was found to induce chromosomal aberrations in various cell systems. Specifically, this occurred in mouse mammary carcinoma cells at concentration of 3.2 mM [94]; in Syrian hamster embryo cells, at 20 mM [95]; in Chinese hamster fibroblasts, at 0.25 mg/mL [31]; and in monkey fetal liver cells and HeLa cells, at 0.265 mg/mL [96]. Despite a wide variety of studies conducted across different organisms and concentrations of nitrites, the panel concluded that there is insufficient evidence to establish a link between dietary nitrites and different types of cancer [17].

Conversely, alkaline elution studies in V79 cells [97] and mouse mammary carcinoma cells [94] showed no evidence of DNA single-strand breaks induced by sodium nitrite treatment.

The ADIs established for potassium and sodium nitrite by the SCF [98] and the JECFA [99] were set at 0–0.06 mg/kg × bw/day and 0–0.07 mg/kg × bw/day, respectively. The available information indicates no in vivo genotoxic potential for either of these substances. Furthermore, exposure to nitrite from its use as a food additive did not exceed the established ADI for the general population, except for a slight exceedance in children [17].

Our results are in agreement with these conclusions: at ADI concentrations, nitrites are safe, but at higher doses than the allowed, they could induce damage in Drosophila.

(2)

Nitrates

Nitrates are considered substances with low toxicity by researchers, but because they are the end product of nitrification, they can accumulate in large quantities [100]. These substances can cause lethal or sublethal effects or even act synergistically with other forms of nitrogen, making it extremely important to study their toxic effects on different species [101], although, on the other hand, Hernández et al. [102] have shown that dietary nitrates improve calcium management.

In vivo toxicity and antitoxicity assays showed a dose-dependent trend at the highest concentration of sodium nitrite; and on the other hand, the highest concentrations of potassium nitrate and sodium nitrate were the only ones that presented a significant toxic and antitoxic effect for Drosophila, with respect to the control. At the genomic level in living individuals, no significant effect was shown on the induction of mutations, although there was also no protection against genotoxin, with the exception of potassium nitrite and sodium nitrite, which showed a mutagenic effect in the combined treatments. Longevity and quality of life assays in our eukaryotic model organism showed very heterogeneous results with respect to the control, with potassium nitrate being the additive that showed a greater dose-dependent improvement in the life extension of flies and potassium nitrite being the one that presented a greater reduction in the quality of life of the treated individuals.

Regarding in vitro studies, both nitrates studied and sodium nitrite were able to induce HL-60 tumor cell death at the maximum concentrations tested. However, if we take into account the dose established as ADI, none of these additives studied presented cytotoxic capacities; on the contrary, they even stimulated the growth of human leukemia cells at such concentrations. There was also no damage at the genomic level in HL-60 cells although nitrates and potassium nitrite induced positive and significant methylation activities with respect to the control.

When food additives are given to organisms in excessive amounts, they can cause toxic reactions. Foods containing potassium nitrate and nitrite in high amounts can react with endogenous amines, forming carcinogens and mutagens [103]. However, when used in small amounts, they also have disadvantages, such as inadequate coloration or insufficient antimicrobial activity and the effects on the final product. In addition, nitrite prevents the growth of Clostridium botulinum and the formation of toxins [104].

Longevity is a measurable trait influenced by many factors, including genetics, genomic regions, environmental conditions, and diet [105]. Mutations affecting genes and stress can modify life expectancy in these genes associated with telomeres [106], which are essential to understand the complex pattern of ageing process in humans [107]. A study on the effects of potassium nitrate supplementation in Drosophila indicated that low dosage (1% and 2%) increased the lifespan of the flies, while a higher dosage (3%) led to a decrease in the survival of the organisms [25].

There are no specific studies on the effect that potassium nitrite and nitrate have on the proliferation of human leukemia cells. However, there are numerous studies on the effects that the consumption of nitrites and nitrates causes in other types of cancer. Van Loon et al. [108] showed that there is no relationship between the intake of nitrites and nitrates and suffering from stomach cancer. Furthermore, studies on nitrate levels in water also showed no association with Hodgkin lymphoma and colorectal cancer [30,109], stomach, or oesophageal cancer [11,29]. These data are associated with our results obtained in the cytotoxicity test since the inhibition of cell growth has been slight on the cytotoxic effects that potassium nitrate and nitrite have.

The current ADI for sodium and potassium nitrate of 3.7 mg/kg × bw/day was established by the SCF [98] and JECFA [62,99] whose available data did not indicate genotoxic potential. Moreover, studies about carcinogenicity in mice and rats were negative [11].

There was some evidence suggesting that nitrate intake from drinking water may be linked to goiter. However, the only study examining the relationship between self-reported hypothyroidism and estimated dietary nitrate intake [110] was deemed inadequate for establishing a reference point for the ADI.

Our in vivo and in vitro results for the food preservatives studied are related to other previous studies carried out where at ADI concentrations, nitrites are safe; however, in excessive concentrations, these additives can be harmful to the model organisms studied and, consequently, potentially harmful to humans.

5. Conclusions

Analyzing the results obtained at the ADI concentration, the only beneficial effects were observed in the toxicity, genotoxicity, antigenotoxicity, and quality of life tests in Drosophila, as well as a neutral effect in the antitoxicity and longevity tests in this in vivo model organism.

If we analyze the in vitro results obtained by these compounds, they all show null or negative effects in the different tests carried out, with the exception of methylation status tests at some specific repetitive sequences for both nitrites and sodium nitrate.

In conclusion, we can say that our tested compounds proved to be safe for Drosophila melanogaster. Regarding nutraceutical potential, sodium nitrate showed beneficial effects for in vivo trials, while potassium nitrite and sodium nitrite showed negative nutraceutical potential for Drosophila. Finally, potassium nitrate showed a slight positive chemopreventive potential in the in vitro assays carried out.

These results, which are in slight disagreement compared to the currently available data supporting the safe consumption of food additives, may be influenced by various factors. These factors include the types of model organisms used, the test conditions, and the range of concentrations tested, among others.

Given the widespread use of different food additives and the varying results from studies, it is essential to conduct ongoing scientific research and thorough evaluations to establish safe dosage levels and usage guidelines for each additive. Additionally, regardless of the food regulations in different countries, it would benefit consumers if food manufacturers considered the findings from research conducted on both in in vivo and in vitro model organisms. This could enhance food quality and improve the well-being of consumers.