B.P. Kuzminov¹, T.S. Zazulyak¹, D.D. Ostapiv², N.E. Yunak¹

¹ Lviv Danylo Halytsky National Medical University, Lviv, Ukraine
² Institute of Animal Biology, National Academy of Sciences, Lviv, Ukraine

RESUME. Goal. To evaluate the cytotoxic effect of different concentrations of lotion containing minoxidil. As a test object, a suspension of spermatozoa was used. Sperm samples were divided into control and experimental ones: with the addition of 5% and 10% lotion containing minoxidil.

Research methods — to determine the survival of sperm (hours) at a temperature of 2–4°C until the cessation of translational movement, respiratory activity — polarographically (ng-atom O₂/min x 0.1 ml of sperm) at a temperature of 38.5°C and regenerative capacity — potentiometrically (mV/min x 0.1 ml of semen), activity of succinate dehydrogenase (SDH; unit/hour x 0.1 ml of semen) and cytochrome oxidase (CCO; unit/hour of 0.1 ml of semen).

Conclusions. It was established that the respiratory activity and regenerative ability of sperm depends on both the dose and the concentration of minoxidil in the lotion.

Key words: minoxidil, alopecia, cytotoxic effect, bull sperm.

Introduction. Androgenetic alopecia is the most common cause of hair loss, affecting at least 50 % of males who have reached the age of 50 years and 70 % of older males [1]. There are many different means of treatment and prevention of alopecia worldwide — from systemic medicines to topical use in the form of lotions [2]. The most effective for stimulating hair growth, both in androgenetic and in other types of alopecia, are drugs that include peripheral vasodilators (potassium channel activators) [3]. Recently, in order to obtain the therapeutic effect, the addition of medicinal active ingredients, in particular, minoxidil began, with the classification of such products as medicinal cosmetics sold in pharmacies and medical centres as the result of consultation with a pharmacist or doctor. This does not take into account the possibility of developing side effects and remote effects.

The data of scientific literature suggest a number of shortcomings in the application of such lotions. Efficiency is observed only for the initial forms of alopecia, and in areas of complete baldness, the restoration of growth is almost not occurring. After stopping the application of the lotion, hair loss may increase again [4].

According to the manufacturer’s instructions, application of the lotion is contraindicated in young people under the age of 18 years and after 65 years of age, development of side effects on the part of sexual potency in males, decreased libido, negative effects on reproductive function and sperm motility may develop.

Since 2004, the testing of cosmetic products on animals has been prohibited in the European Union [5]. Alternative study methods using germ cell motility as a test object are common. The proof is that it is suitable for toxicological studies with a high correlation of results compared to classical toxicological methods using animals [6].

Suspension of bull sperm cells, which is widely used in the practice of artificial insemination and is standardized is highly susceptibility to toxic substances. The advantages of this method have allowed its expansion during toxicological studies in medicine and veterinary medicine.

Objective. Determine the presence of cytotoxic effects of various concentrations of the minoxidil-containing lotion by the duration of survival of the sperm cells and the intensity of the oxidative processes in the bull sperm.

Materials and methods. Ejaculates (n = 3) of 1.83 ± 0.14 mL in volume, the concentration of 0.7 ± 0.10 × 10⁹ cells/mL and activity of 7.4 ± 2.37 points were selected for the study. Sperm was divided into parts: one part was used to determine respiratory and recovery ability of sperm ex tempore in phosphate-saline buffer (NaCl — 0.8 g, KCl — 0.02 g, Na₂HPO₄ — 0.11 g, KH₂PO₄ — 0.02 g, MgCl₂ — 0.01 g, H₂O to 100 mL), and the other part — to study the activity of mitochondrial enzymes and survival of sperm cells in lactose-yolk-glycerol diluent. In this case, a control sample was isolated without addition and test samples — with the addition of 5 % and 10 % minoxidil-containing lotion, at doses of 0.10 mL, 0.05 mL and 0.01 mL per 1 mL of diluted sperm.

The following parameters were determined: survival of the sperm cells (h) at 2–4 °C before stopping the translational movement, respiratory activity — by polarography (ng-atom O₂/min × 0.1 mL sperm) at 38.5 °C [8] and a recovery ability — by potentiometry (mV/min × 0.1 mL sperm) [9], activity of succinate dehydrogenase (SDH; U/h × 0.1 mL sperm) and cytochrome oxidase (CHO; U/h × 0.1 mL sperm) [10, 11]. Statistical analysis of digital material was conducted by M. O. Plokhinskyi [12].

Results and their discussion. It has been established that the respiratory activity and the recovery ability of the sperm depend on both the dose and the concentration of minoxidil in the lotion. Thus, the addition of 0.01 mL of 5 % lotion to the polarograph well leads to 2.5 times (p < 0.05) inhibition of oxygen intake and 4.0 times (p < 0.01) inhibition of transportation of protons (electrons) to extracellular space (Table 1).

Table 1

Respiratory activity and recovery ability of sperm under exposure to lotion, n = 3; M ± m

*Note: Difference is statistically probable compared to the control: * р < 0.05; ** р < 0.01; *** р < 0.001

Increasing the dose to 0.05 mL still reduces respiratory activity by 43.4 %, which value is 4.3 times lower than control. By contrast, the recovery ability under the specified dose of lotion increases to 3.0 ± 0.28 mV/min × 0.1 mL (66.7 %; p < 0.01), but remains lower by 25.0 % compared to control. Increasing in the amount of 5 % lotion to 0.10 mL reduces respiratory activity by 35.3 % (p > 0.05), compared to a dose of 0.05 mL. At the same time, the recovery ability of sperm is inhibited by 40.0 %.

Similar changes were found in the study of the effect of similar doses of 10 % lotion on respiratory activity and recovery ability of sperm. At 0.01 mL of the specified concentration of lotion, respiratory activity and recovery ability are in 3.3 times (p < 0.01) and 2.8 times (p < 0.01) lower than in the control. A dose increase of 10 % lotion to 0.05 mL and more inhibits the consumption of oxygen by sperm 12,3 to 26,6 times (p < 0.01–0.001) compared to the control. The recovery ability of 0.05 mL of 10 % lotion is lower by 25.0 % and of 0.10 mL — by 65.0 % (p < 0.05) than that of the control.

The evaluation of the activity of the enzymes of the sperm mitochondria revealed that the action of 0.01 mL of 5 % lotion increases SDH by 36 % (p < 0.05), and CHO does not change and is within the control (33.3 ± 2.72 U/h × 0.1 mL; Table 2).

Table 2

Activity of mitochondrial enzymes and survival of sperm cells under exposure to lotion, n = 3; M ± m

*Note: Difference is statistically probable compared to the control: * р < 0.05; ** р < 0.01; *** р < 0.001

Increase of 5 % lotion to 0.05 mL leads to inhibition of enzymatic activity, respectively, SDH by 35.7 % and CHO by 25.2 %, as compared to control. At the maximum dose (0.10 mL of 5 % lotion) the activity of enzymes reaches the minimum value: SDH — 0.2 ± 0.03 and CHO — 18.3 ± 1.36 U/h×0.1 mL, which is respectively lower by 99.2 (p < 0.001) and 45.3 % (p < 0.01), as compared to control.

Similar changes in the activity of the enzymes of the sperm mitochondria have been established under the use of 10 % lotion. However, under exposure to 0.01  mL of lotion, the activity of SDH and CHO is at the control level and amounts to 21.7 ± 1.36 and 31.7 ± 3.60 U/h×0.1 mL, respectively. A dose increase to 0.05 mL leads to inhibition of enzymatic activity by almost the same value of 60.2–64.6 % (p < 0.01). Under the maximum dose of 10 % lotion (0.10 mL), the activity of SDH and activity of CHO is lower by 99.6 % and 65.0 % than that of the control.

Reduction in the intensity of oxidative processes and the activity of mitochondrial enzymes under increased content of lotion in diluted sperm leads to changes in the survival time of sperm cells. In particular, under exposure to 5 % lotion in the amount up to 0.05 mL, the value of physiological parameter is almost unchanged and is within the control (104.0–106.7 h), and in the amount of 0.1 mL the survival time of sperm cells is reduced by 61.9 % (p < 0.01) compared to control.

Addition of 10 % lotion in increasing doses leads to a decrease in the survival of sperm cells. For example, under exposure to 0.01 mL, the value of the physiological parameter is lower by 32 hours (28.6 %, p < 0.05) than in the control. A dose increase to 0.05 mL reduces the survival of sperm cells by 54.8 % (p < 0.001) and to 0.01 mL — by 70.4 % (p < 0.001), compared to control.

From the analysis of the dependence of oxidative processes in sperm and survival of sperm cells following addition of increasing doses of the lotion, it follows that the active ingredient inhibits respiratory activity, and recovery ability, and activity of mitochondrial enzymes of the germ cells, which leads to a decrease in the survival of sperm cells. At the same time, changes in the studied parameters depend on the concentration and dose of the active ingredient in diluted sperm. The strength of effect of increasing content of 5 % and 10 % lotion on the respiratory activity and the activity of mitochondrial enzymes is negative and strong (η² = 0.845 and 0.874 and η² = 0.924 and 0.917, respectively), and on the recovery ability — of moderate strength (η² = 0.633 and 0.462). Effect of increasing lotion doses on the survival of sperm cells is negative, and the strength of the effect increases with an increase in the percentage content of minoxidil (5 % — η² = 0.855 and 10 % — η² = 0.942, respectively).

Conclusion

1. The cytotoxic action of the minoxidil-containing lotion depends on its concentration and dose and is characterized by impairment of the oxidative processes of the sperm, manifested by reduced oxygen intake and decreased recovery activity.
2. In substantiating the evaluation of the potential and actual risk of the development of remote effects of the minoxidil-containing lotion, it is necessary to conduct a gonadotoxicity study in laboratory animals.

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Надійшла до редакції 27.02.2019р.