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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access   Full Text Article                                                                                                                                                     Research Article

Effect of pH on Toxicity of Zinc Sulphate Hydrate Solutions Using the Spirotox Test

Quynh Thi Ngoc Hoang *, Gleb V. Petrov, Alena M. Koldina^*, Anton V. Syroeshkin

Department of Pharmaceutical and Toxicological Chemistry, Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho - Maklaya St., Moscow, 117198, Russian Federation. 

INTRODUCTION 

Zinc is essential for the vital activity of organisms. It is involved in synthesizing and activating various enzymes, regulating cell growth, and supporting growth and development in animals and humans, among other physiological functions ^1,2. Zinc is a crucial trace element for maintaining and developing immune cells in both the innate and adaptive immune systems. Its deficiency can lead to a decrease in the body's immune response, an increased risk of inflammation, and in some cases, even fatal outcome ³. Zinc also regulates and stimulates the proliferation, differentiation, and secretion of mammary glands during lactation ⁴. Additionally, it can be used in combination with other treatments for diabetes ⁵.

Zinc is an immunomodulator that has multiple effects against a wide range of viral species, particularly RNA viruses. When used in combination with antiviral drugs at low concentrations, zinc inhibits SARS-CoV-2 replication in vitro ⁶. Mechanism of action of zinc in these processes achieved by inhibiting the elongation stage of RNA transcription. Furthermore, zinc can enhance antiviral immunity by upregulating IFN-α ^7,8. Therefore, the multifunctionality of zinc is of interest for research in the field of pharmaceutical chemistry.

Toxicity evaluation is a significant safety factor of an active pharmaceutical ingredient. This article proposes a method for analyzing the toxicity of heavy metals ions, such as Zn²⁺, using the Spirotox test and the cell biosensor S. ambiguum ⁹. It is highly sensitive to a wide range of chemicals, including drugs containing heavy metals ions, antimycotics, adrenoblockers, antiarrhythmics, and neuroleptics ¹⁰. S. ambiguum can be stored for several weeks at temperatures ranging from 5 to 28 °C and in a pH range of 5 to 8. This method is highly effective in assessing the toxicity/pH ratio ¹⁰. Previous studies ^10,11 have established the dependence of toxicity on the pH value of the solution using the Spirotox test for antidepressants and nitrophenol. Studies have tested the influence of pH on the toxicity of heavy metal drugs on fish, vertebrates ^12,13, and microalgae (Chlorella species, Pseudokirchneriella subcapitata) ^14–16. However, the relationship between solution pH and the toxicity of zinc ions in pharmaceutical substances with varying degrees of hydrating has not yet been tested on the model S. ambiguum.

The objective of this study is to investigate the impact of pH levels ranging from 5.0 to 6.5 on the toxicity of aqueous solutions of various forms of zinc sulphate using the S. ambiguum. Additionally, the study aims to compare the activation energies at different pH values, which is crucial in establishing quality standards for zinc-containing pharmaceuticals.

MATERIALS AND METHODS 

The toxicity of a 0.01 M solution of ZnSO₄⋅H₂O and ZnSO₄⋅7H₂O was investigated using a Spirostomum ambigua cell biosensor (Spirotox test). The test setup comprised deionized water ([D/H] = 140 ppm, purified using Milli-Q, Merck Millipore, USA), a thermostatic 5-cell plate with a set temperature ranging from 22°C to 28°C in 2°C increments (Lauda Alpha 6, Germany), a binocular to observe infusoria behavior, and low-power lamps (10 W). The solution's pH was adjusted using dilute sulfuric acid (H₂SO₄) or potassium hydroxide (KOH) and a laboratory pH meter PB-11 (Sartorius, Germany). Toxicity tests were conducted at five pH values that correspond to normal living conditions of S. ambiguum. The results were processed using Origin Pro 9.1 software (OriginLab, USA) and presented as mean ± SD (n=5).

RESULTS

The interaction of S. ambiguum with the solution under study is determined by the rapid formation of an intermediate state, which undergoes a slow transition to a stationary state according to Arrhenius kinetics.

Cell death is defined as a transition to a state of either immobility or membrane rupture. When cellular transitions are induced by ligands, the temperature dependence of S. ambiguum lifetime can be linearized in Arrhenius coordinates, which follow the form of the Arrhenius equation: 

where: k is the rate constant of the process; A is the pre-exponential multiplier; R is the universal gas constant; T is the absolute temperature, K.

Zinc is an essential element with a therapeutic function. There are areas of both zinc deficiency and zinc surplus. It has been demonstrated that when the initial solutions of zinc sulphate monohydrate were diluted tenfold, the lifetime of S. ambiguum significantly increased (Fig. 1). Therefore, a solution concentration of 0.01 M was deemed most suitable for the study. Higher concentrations are required to optimize the experiment time ¹⁷.

Figure 1: Dependence of S. ambiguum lifetime on temperature at different zinc concentrations in ZnSO₄.H₂O solution (n=5)

Figure 2 (A, B) illustrates the dependence of the activation energy of ligand-induced death of S. ambiguum on the pH in ZnSO₄⋅7H₂O and ZnSO₄⋅H₂O solutions at a zinc concentration of 0.01 M.

Figure 2: Dependence of activation energy of ligand-induced death of S.ambiguum on pH, where A - ZnSO₄.7H₂O, B - ZnSO₄.H₂O (mean±SD, n=5)

It was demonstrated that, at the same zinc dosage, the survival time of infusoria decreased gradually as the pH increased. The observed Ea is likely dependent on the sample preparation history. Furthermore, the influence of Zn ions on the formation of ligand-dependent density inhomogeneities, also known as “bubstones”, should be considered ^18,19. When the solutions had equal zinc concentrations, the solution prepared from monohydrate showed greater toxicity compared to the solution prepared from zinc sulphate heptahydrate. The pH increased from 5.0 to 6.5, the Ea gradually decreased, indicating an increase in the environment's toxicity for infusoria.

DISCUSSION

It is widely acknowledged that any element can have toxic effects when it reaches certain concentrations in the organism. The most essential elements are included in the composition of dietary supplements, and their quantitative content is not standardized, it is critical to control these preparations for toxicity. Therefore, the daily dose of zinc preparations for an adult range from 10 to 100 mg ²⁰.

The role of pH in the metabolism of zinc sulphate hydrates is significant. The major forms of zinc in solution change from  ,  ,  and   to the readily soluble ionic form   as pH decreases ²¹. The ratio of OH^- to   concentration gradually increases as the pH of the solution increases ²². Multiple studies have demonstrated that metal toxicity in fish and invertebrates increases as pH decreases, due to the prevalence of free metal ions ^12,13. In our experimental setup, the lifespan of S. ambiguum decreased as pH levels approached neutrality. The authors demonstrated an increase in metal toxicity with increasing pH in different studies ^14–16. This effect was due to a decrease in competition between metal ions and H⁺ on the membrane surface. According to research ²³, a decrease in the concentration of H⁺ in solution is believed to be linked to the death of infusoria. This decrease causes an increase in the permeability of the S.ambiguum cell membrane, resulting in rapid swelling and cell rupture ²³.

The toxicity of zinc sulfate hydrate solution for S. ambiguum is affected by pH value. An increase in pH from 4.8 to 6.5 resulted in a 2.6-fold decrease an Ea according to the law of Arrhenius kinetics for zinc sulfate heptahydrate, indicating an increase in toxicity. The article considers a control method using the S. ambiguum to manage the toxicity of pharmaceutical substances containing zinc sulfate mono- and heptahydrates. 

CONCLUSION

In the present study, the biological activity for the Spirotox test was shown to be altered by increasing the pH of solutions of various hydrated forms of zinc sulphate. S. ambiguum is known to be stable over a wide pH range. Therefore, the toxic effect on infusoria is not caused by a change in the acidity index of environment, but by the effect of the xenobiotic on their cellular wall. The Spirotox test can be used for the control of the toxicity of zinc sulphate pharmaceutics.

Acknowledgment

This paper has been supported by the RUDN University Strategic Academic Leadership program. 

Conflict of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

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