Dose-dependent effect of cadaverine on hydroxyl radicals’ production by human peripheral blood leukocytes

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Introduction

Currently, there is growing interest in the role of polyamines in intercellular communication, especially in the context of microbial communities and inflammation development [1], due to the accumulation of these compounds at the focus of inflammation and their unique ability to influence the cellular processes of eukaryotic cells without the involvement of specific receptors [2; 3].

It is known that polyamines such as cadaverine and putrescine affect gene expression, activate signaling cascades, and modulate intercellular signals, making them significant objects for research [4–6]. The main mechanisms of action of polyamines are believed to be their penetration into the cell through secondary active transport, modification of protein molecules, binding to DNA and RNA molecules [5; 7]. Cadaverine, which belongs to the aliphatic diamines in terms of its chemical structure, attracts attention among the polyamine family [6]. It has been shown that various factors, including nutrient deficiency and exposure to antimicrobial and other substances present in large amounts at the focus of inflammation, activate the synthesis of cadaverine by some microorganisms (Escherichia coli, Salmonella Typhimurium, Vibrio cholerae, etc.) [8–10].

As is known, the formation of reactive oxygen species by leukocytes, such as hydrogen peroxide, superoxide anion radical, singlet oxygen, hydroxyl radical and hypochlorite, is a key factor in the process of destruction of ingested objects [11; 12]. A disruption of this process leads to incomplete phagocytosis and generally contributes to the ineffectiveness of the entire cellular component of the immune response [13]. In previous studies on polyamines, a stimulating effect of cadaverine on the radical-producing activity of leukocytes was revealed [4; 14]. At the same time, the enzyme diamine oxidase of leukocytes performs anti-mediator anti-inflammatory functions by inactivating histamine and other diamines, including cadaverine and putrescine [19]. This can create a favorable environment for microorganisms, since their structures may be protected by an excess of polyamines, which, in turn, stimulate the anti-mediator activity of diamine oxidase [20; 21]. As a result, there is a decrease in inflammation activity [7; 22], which is also confirmed by in vivo studies [23; 24].

Currently, it is of interest to study the dose-dependent effect of this polyamine.

The aim of the study is to assess changes in the generation of hydroxyl radicals by leukocytes under the influence of cadaverine at concentrations of 1; 5 and 25 mmol/L.

Materials and methods

Peripheral venous blood samples were obtained from 40 apparently healthy donors. To assess the production of hydroxyl radicals, a luminol-dependent chemiluminescence (LDCL) reaction was performed with blood leukocytes [15], isolated by sedimentation using 0,1 % methylcellulose (USA), followed by incubation with cadaverine at concentrations of 1; 5 and 25 mmol/L for 60 minutes at 37 °C. When setting up the LDCL reaction, we used
2·10^–4M luminol sodium salt (Sigma, USA), cells pretreated with cadaverine (25·106/ml). The measurement was carried out on a Luminoskan Ascent® Thermo Labsystems luminometer (USA) for 180 minutes. For statistical analysis, the integral chemiluminescence indicator for the entire measurement period (RLU) was used. The time to reach the maximum of light flashes in minutes, the intensity of the utmost glow, and the area under the luminescence extinction curve were calculated.

Statistical data processing was carried out using the Statistica 7.0 package. The Shapiro – Wilk test was used to check for normality of distribution. In case of a distribution close to normal, Student's t-test was used, otherwise Mann – Whitney U test was applied to assess significance of differences. The threshold significance level was set at p < 0.05. Results are presented as mean and its error (M ± m).

Results and discussion

It was found that incubation of cells with cadaverine significantly reduces their time to reach the maximum generation of hydroxyl radicals (Fig. 1), which was 19.3 ± 0.8 minutes at a cadaverine concentration of 1 mmol/L (p = 0.009 compared to samples with spontaneous reaction); 32.3 ± 4.8 and 19.0 ± 0.8 min at 5 and 25 mmol/l, respectively (p = 0.09 and 0.02 compared to samples with spontaneous reaction).

Fig. 1. Time for the LDCL reaction to reach maximum when cells are incubated with cadaverine, min

The maximum indicator chemiluminescence index for the entire measurement period at a cadaverine concentration of 1 mmol/L was 0.081 ± 0.004 RLU, at 5 mmol/l was 0.130 ± 0.013 RLU, and at 25 mmol/l was 0.083 ± 0.022 RLU (p = 0.008; 0.009 and 0.004 compared to samples with spontaneous reaction). As can be seen from Fig. 2, cadaverine significantly enhances the intensity of the LDCL reaction of leukocytes in healthy donors, with the greatest stimulating effect recorded for a polyamine concentration of 5 mmol/l (p = 0.009 compared to samples with spontaneous reaction, p = 0.007 and 0.010 compared to samples with cadaverine 1 and 25 mmol/l, respectively).

Fig. 2. The maximum intensity of the LDCL reaction when leukocytes are incubated with cadaverine, RLU

A similar effect was also found when calculating the area under the LDCL reaction curve (Fig. 3). Cadaverine increases this indicator, and at a concentration of 5 mmol/l, it is most significant – 20.89 ± 3.00 vs. 0.86 ± 0.07 conventional units for spontaneous reaction (p = 0.001).

Fig. 3. The area under the curve of the LDCL reaction when leukocytes are incubated with cadaverine., c. u.

It is known that the synthesis of polyamines by bacteria in the inflammation focus increases. At the same time, the gradual accumulation of cadaverine probably has a stimulating effect on the generation of radicals by leukocytes, which manifests itself in an increase in the rate of their production, as evidenced by the acceleration of the time to reach the peak, as well as the accumulation of active forms, as evidenced by an increase in the area under the curve. However, with a further increase in the concentration of polyamines, cadaverine plays the role of a «scavenger» of radicals, reducing their content, which is expressed in a decrease in the area under the curve in the LDCL reaction.

The increase in radical production noted in this study, induced by pretreatment of neutrophils and monocytes with cadaverine, indicates the possible influence of this compound on the mechanisms of hydroxyl radical formation, as well as the activity of diamine oxidase in leukocytes [16]. This enzyme catalyzes the conversion of diamines into aminoaldehyde, which is accompanied by the release of hydrogen peroxide, followed by interaction with luminol present in the reaction mixture [13]. It is worth noting that the activity of diamine oxidase increases almost instantly after the initiation of phagocytic activity [17; 18].

Conclusions

1. Pre-incubation of leukocytes with cadaverine increases their production of hydroxyl radicals.
2. The dependence of hydroxyl radical production by leukocytes on cadaverine concentration is shown. At the same time, the average concentration of cadaverine has a more pronounced effect.

About the authors

I. A. Morozov

Author for correspondence.
Email: Lonny8@yandex.ru
ORCID iD: 0000-0003-4233-3711

Postgraduate Student of the Department of Microbiology and Virology

References

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2. Fig. 1. Time for the LDCL reaction to reach maximum when cells are incubated with cadaverine, min

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3. Fig. 2. The maximum intensity of the LDCL reaction when leukocytes are incubated with cadaverine, RLU

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4. Fig. 3. The area under the curve of the LDCL reaction when leukocytes are incubated with cadaverine., c. u.

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