1. Introduction

In recent years, the use of synthetic food additives, particularly food colorants, has significantly increased in the food industry. Among them, titanium dioxide (E-171) and aluminum powder (E-173) are widely used to enhance the visual appeal of various food products such as confectioneries, dairy goods, and beverages. Despite their technological advantages, numerous studies have indicated that these substances may exert toxic effects on different organs and tissues of the human body, including the endocrine system [1,2,3,4,5,9,12]. The adrenal glands, especially the adrenal cortex, play a vital role in maintaining homeostasis through the secretion of corticosteroids, mineralocorticoids, and sex hormones. Any disruption in the structure or function of the adrenal cortex may lead to metabolic disorders, impaired stress response, and hormonal imbalance. Therefore, studying the morphofunctional changes in the adrenal cortex under the influence of food dyes such as E-171 and E-173 is of great scientific and practical importance. Furthermore, understanding the potential corrective mechanisms, including the use of antioxidants and natural protective agents, can contribute to developing effective strategies to reduce the harmful effects of these additives. This research aims to analyze the structural and functional alterations in the adrenal cortex caused by titanium dioxide and aluminum-based colorants, as well as to assess the possibilities of their correction through biologically active substances [6,7,8,9,10,11].

2. Methods

The study was conducted using a combination of biochemical, histological, immunological, and analytical techniques to comprehensively assess the morphofunctional state of the adrenal cortex under the influence of food dyes E-171 and E-173. Histological examinations were performed using standard staining methods, including Masson’s trichrome, hematoxylin-eosin, and Van Gieson techniques to evaluate the structural organization and connective tissue components of the adrenal cortex. Immunological analysis was carried out using the ELISA (Enzyme-Linked Immunosorbent Assay) method to determine specific biochemical markers. For quantitative assessment, a nuclear physics-based analytical method — electron activation analysis — was employed to measure the concentration of certain elements within the tissue samples. Additionally, morphometric, immunohistochemical, and statistical methods were utilized to analyze and interpret the obtained data with high accuracy.

3. Results

Experimental exposure to food dyes E-171 (titanium dioxide) and E-173 (aluminum-based compounds) resulted in pronounced morphofunctional alterations in the adrenal cortex of laboratory animals compared with the control group. The observed changes were characterized by structural disorganization of cortical zones, disruption of cellular architecture, and significant shifts in morphometric parameters, indicating impaired steroidogenic activity.

Quantitative morphometric analysis revealed statistically significant deviations (p < 0.05) in cell size, nuclear dimensions, cytoplasmic area, and nuclear–cytoplasmic ratio across all cortical zones, with the most prominent alterations detected in the zona fasciculata and zona reticularis.

In animals exposed to E-171 and E-173, the zona glomerulosa demonstrated a reduction in average cell area and nuclear diameter compared to controls. Cellular cords appeared fragmented and less organized, with increased intercellular spaces. These changes suggest a decline in functional activity of glomerular cells and possible impairment of aldosterone synthesis. Morphometric data indicated a moderate increase in nuclear–cytoplasmic ratio, reflecting cytoplasmic depletion and reduced biosynthetic capacity. Combined exposure to both dyes intensified these alterations, pointing to a synergistic toxic effect on mineralocorticoid-producing cells. The zona fasciculata exhibited the most pronounced morphofunctional disturbances. Exposure to food dyes led to a significant decrease in cytoplasmic volume, accompanied by vacuolization and irregular cellular contours. Although a compensatory enlargement of nuclei was observed, total cell size remained markedly reduced compared to the control group. The nuclear–cytoplasmic ratio increased significantly (p < 0.01), indicating persistent metabolic stress and impaired glucocorticoid synthesis. These findings suggest that E-171 and E-173 disrupt enzymatic systems involved in steroidogenesis, particularly those responsible for cortisol production.

In the zona reticularis, exposure to food dyes resulted in altered cellular arrangement and loosening of the reticular architecture. Cells demonstrated reduced cytoplasmic area and decreased staining intensity, indicative of suppressed androgen synthesis. Despite partial recovery of nuclear dimensions in correction groups, cytoplasmic restoration remained incomplete. The nuclear–cytoplasmic ratio stabilized only partially, suggesting ongoing metabolic tension and insufficient normalization of steroidogenic function.

Corrective treatment led to partial normalization of adrenal cortex structure. Morphometric indicators showed a tendency toward recovery, including increased cell and cytoplasmic areas, as well as improved zonal organization. However, most parameters did not reach control values, particularly in the zona fasciculata and zona reticularis. Animals receiving corrective therapy demonstrated reduced severity of dystrophic changes and improved cellular integrity, supporting the protective and restorative potential of the applied correction strategy. Nevertheless, residual morphofunctional alterations indicate that prolonged or combined exposure to food dyes exerts lasting effects on adrenal cortical cells.

Violin plots and comparative diagrams generated using Python software confirmed the heterogeneity of cellular responses across experimental groups. Statistical analysis validated the reliability of observed differences, emphasizing the dose-dependent and zone-specific impact of E-171 and E-173 on adrenal cortical morphology and function.

4. Discussion

The findings of the present study clearly demonstrate that food dyes E-171 (titanium dioxide) and E-173 (aluminum-based compounds) exert a pronounced adverse effect on the morphofunctional organization of the adrenal cortex. The detected alterations reflect a complex disruption of cortical zonation, cellular metabolism, and steroidogenic activity, indicating that prolonged exposure to these compounds compromises endocrine homeostasis. Compared with the control group, all experimental groups exhibited a significant reduction in cell and cytoplasmic dimensions accompanied by an increased nuclear–cytoplasmic ratio. This pattern suggests that adaptive–compensatory mechanisms activated in adrenal cortical cells remain insufficient under conditions of sustained toxic exposure. The predominance of nuclear preservation over cytoplasmic restoration reflects a shift toward metabolic stress and reduced biosynthetic capacity.

In the zona glomerulosa, fragmentation of cellular cords and cytoplasmic depletion indicate suppressed functional activity of mineralocorticoid-producing cells. The elevated nuclear–cytoplasmic ratio reflects impaired enzymatic processes involved in aldosterone biosynthesis. The combined exposure to E-171 and E-173 intensified these changes, supporting the existence of a synergistic toxic effect that disrupts the structural integrity of the glomerular zone.

The zona fasciculata showed the most pronounced morphofunctional disturbances, highlighting its heightened sensitivity to toxic agents. Although nuclear enlargement appeared to represent a compensatory response, the persistent reduction in cytoplasmic volume suggests severe impairment of organelles critical for steroidogenesis, particularly mitochondria and smooth endoplasmic reticulum. These changes provide a morphological basis for decreased glucocorticoid synthesis and sustained metabolic tension within fasciculata cells.

In the zona reticularis, loosening of the reticular architecture and diminished cytoplasmic density point to suppressed androgen production. Partial normalization of nuclear dimensions in correction groups, coupled with incomplete restoration of the cytoplasm, indicates that metabolic stress persists despite adaptive cellular responses. The failure of the nuclear–cytoplasmic ratio to fully normalize underscores ongoing functional instability.

The adverse effects of E-171 and E-173 may be attributed to their ability to induce oxidative stress, disrupt membrane stability, and inhibit key enzymes involved in steroid hormone biosynthesis. Titanium and aluminum compounds are known to impair mitochondrial function, leading to decreased ATP production and compromised cellular energy balance. As a result, the biosynthetic potential of adrenal cortical cells becomes markedly reduced. The consistent increase in the nuclear–cytoplasmic ratio observed across cortical zones suggests a shift toward catabolic processes, reflecting suppressed protein and steroid hormone synthesis. Such changes indicate that adrenal cortical cells fail to achieve complete adaptation under prolonged toxic exposure.

Corrective treatment resulted in partial restoration of adrenal cortical structure and morphometric parameters; however, most indicators did not reach control levels. This incomplete recovery highlights the depth and persistence of dye-induced cellular damage. The slow regeneration of the cytoplasmic compartment suggests that enzymatic and organelle systems involved in steroidogenesis require prolonged recovery periods or more intensive corrective strategies.

Nevertheless, the reduction in dystrophic changes and partial normalization of cortical architecture observed in correction groups confirm the cytoprotective and adaptive potential of the applied intervention. The results of this study provide strong morphofunctional evidence that commonly used food dyes can adversely affect the adrenal cortex, thereby disrupting endocrine regulation. These findings underscore the importance of reassessing the long-term safety of food additives and developing effective strategies to mitigate their toxic impact on endocrine organs.

5. Conclusions

The present study demonstrates that food dyes E-171 (titanium dioxide) and E-173 (aluminum-based compounds) induce significant morphofunctional alterations in the adrenal cortex. Exposure to these additives disrupts the structural organization of cortical zones, alters cellular architecture, and adversely affects key morphometric parameters, indicating impaired steroidogenic function.

The most pronounced changes were observed in the zona fasciculata and zona reticularis, where persistent reductions in cytoplasmic volume and increases in the nuclear–cytoplasmic ratio reflected sustained metabolic stress and suppression of glucocorticoid and androgen synthesis. Although partial nuclear compensation was evident, incomplete cytoplasmic recovery suggests deep damage to enzymatic systems and organelles essential for steroid hormone biosynthesis.

Combined exposure to E-171 and E-173 intensified morphofunctional disturbances, supporting the presence of a synergistic toxic effect on adrenal cortical cells. These findings highlight the heightened vulnerability of adrenal tissue to prolonged exposure to food dye components, particularly those capable of inducing oxidative stress and mitochondrial dysfunction.

Corrective interventions led to partial restoration of adrenal cortical structure and improvement of morphometric indicators; however, most parameters failed to return to control values. This incomplete recovery underscores the persistent nature of dye-induced cellular damage and suggests that longer or more targeted corrective strategies may be required to fully normalize adrenal function.

In summary, the data provide compelling evidence that chronic exposure to E-171 and E-173 poses a potential risk to adrenal cortical integrity and endocrine regulation. These findings emphasize the necessity for further experimental and clinical studies to assess the long-term endocrine safety of food dyes and to develop effective preventive and corrective approaches.