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Review Article

Histological and Physiological Studies on Adult Female Albino Rats Treated with Paraphenylenediamine

M
Mai Almosaibih1
A
Afrah ALrashidi1,*
1Department of Biological Science, College of Science, University of Jeddah, Saudi Arabia.

ABSTRACT

Background: Women are exposed to Paraphenylenediamine (PPD) during hair dyeing in a variety of Arab nations, PPD may damage internal organs and result in death. The current study intends to assess the histological and physiological studies on female albino rats treated with Paraphenylenediamine.

Methods: 24 females rats were used in the study and divided into four groups each with 6 rats: group1: control female group, rats will be treated, orally with distilled water for 8 weeks, group 2 treated female group, rats will receive 10 mg/kg B.W of PPD in 1 ml distilled water once daily for 8 weeks by oral gavage, group 3: withdrawal control female group, rats receive distilled water for 8 weeks and left untreated for two weeks after stopping treatment, group 4: withdrawal treated female group, rats will receive 10 mg/kg B.W of PPD for 8weeks and left untreated for two weeks after stopping treatment.

Result: It was observed on microscopic examination that orally applied PPD solution affects histology of rat ovary marker reduction in ovarian follicle numbers and degeneration of interstitial cells and some follicles, with observable modifications in physiological processes in hormones, In the treated female group, the serum levels of progesterone, estrogen and FSH hormones significantly increased when compared with the control female group. It was concluded from the results that orally applied PPD solution induces histomorphology and physiological changes in the ovary of rats with 10 mg/kg dose.

INTRODUCTION

Paraphenylenediamine (PPD) is an aniline-related aromatic diamine that comes in the form of pinkish gray crystalline lumps with a high oxidation content (Eissa et al., 2021). It is employed as a photographic developing agent, an azo dye intermediate, an antioxidant and a rubber vulcanization accelerator, PPD is added to henna to speed up the dying and drying process, intensify and darken the color, improve the tattoo’s design pattern and make it last longer (Elhelaly and Shaker, 2014). Young adults and teenagers are gaining popular with temporary “black henna tattoos. (Cascão, 2021). PPD is present in most permanent hair dyes (Venkatesan et al., 2021), PPD can also be found as an antioxidant in rubber compounds as well as textile or fur dyes, photographic developing agents, dark-colored cosmetics, temporary tattoos, photocopying and printing inks, black rubber, oils, greases and gasoline (Elmanfe et al., 2022). it is also a component used in the production of polymers, aramid fibers, textile colors and dyes. No matter the brand, almost all natural, herbal, synthetic, or ammonia-free hair dyes include PPD (Brito, 2017).
       
When applied topically or consumed, parapheny lenediamine can have local and systemic hazardous effects (Misirlioglu et al., 2022). Unfortunately, women are exposed to PPD during hair dying because PPD-containing hair dyes are far less expensive than other hair colors on the market (Mohamed et al., 2023). Hair dye has the most hazardous harmful consequences when it is absorbed through the skin, ingested, or inhaled. Depending on the dose and concentration of PPD, it might cause internal organ damage and death (Malik et al., 2016). Exposure to PPD occurs through skin, accidental ingestion, or inhalation of particles from hair dye formulation during dyeing. Acute exposure to high levels of Para phenylene diamine may cause severe dermatitis, asthma, renal failure, edema of face, neck and larynx. Cases of rhabdoms, acute tubular necrosis with acute renal failure and hepatic failure have been reported (Hummdi, 2012). Decrease in hemoglobin leading to anemia due to hemolytic effect of Paraphenylenediamine on red blood cells has been noticed in rats that received sub lethal doses of Paraphenylenediamine (Malik et al., 2016).
       
Bandrowski’s base, an extremely poisonous molecule produced by PPD metabolism (Amin et al., 2021). When PPD is manufactured or used, workers may be exposed to it. Exposure can happen by inhalation, skin contact, eye contact and/or ingestion (Elmanfe et al., 2022). So, the current study aims to evaluate the effect of PPD on female reproductive system.
       
In 1924, a hairdresser reported the first incidence of PPD toxicity due to exposure from PPD dye processing (Waggas, 2011). Since it has been discovered that cosmetics can have both local and systemic effects, they have attracted a lot of attention (de Groot, 2013). Contrary to popular perception, henna tattoos can cause allergic responses, some of which can be quite severe. PPD is one of the substances used in henna tattoos. PPD, a severe contact sensitizer, can be found in black henna tattoos and hair dyes (Misirlioglu et al., 2022). In oxidative hair dyes, paraphenylenediamine (PPD) is one of the most common compounds utilized. However, its use has been linked to negative health impacts (Venkatesan et al., 2021).
    
Due to research showing that cosmetic substances possess both local and systemic effects, there was much of interest in these substances in the past several years. PPD speeds up the coloring process when used in hair dyes (Ibrahim and Maguid, 2016). There is ample evidence of paraphenylenediamine’s effects on the liver, pancreas, kidneys and heart; however, the effects on the female reproductive system are less established. This study was designed to investigate the histological and physiological effects of paraphenylenediamine on the ovary of rats.

MATERIALS AND METHODS

Chemicals
 
The teste chemical paraphenylenediamine (PPD), CAS No (106-50-3) was purchased from Sigma Chemical Company.
 
Animals experimental design
 
A total of 24 adult female rats, (body weight 160±200 g), (n=6) was used in this study. the rat was from the experimental animal unit of faculty of pharmacy. King Abdul-Aziz University, Jeddah, Saudi Arabia (2022). Rats were adjusted to the laboratory conditions for one week before the activation of the experiments. The animals were housed in standard spotless plastic cages and kept in controlled laboratory status of temperature (20±1°C), humidity (65%) and 12 h light: dark cycle. The animals had free line tap water and are ad libitum on normal commercial chow diet.
 
The rats were divided into four groups (n=6):
 
Control group
 
Female rats were treated orally with distilled water.
 
Treated group
 
Experimental group rats were administered 10 mg/kg B.W of PPD in 1 ml distilled water once daily for 8 weeks by oral gavage.
 
Withdrawal control group
 
Female rats of control group fed distilled water for 8 weeks and left untreated for two weeks after stopping treatment.
 
Withdrawal treated group
 
Female rats fed 10 mg/kg B.W of PPD for 8weeks were left untreated for two weeks after stopping treatment.
 
Ethics approval for animal experimentation
 
The King Abdul-Aziz University faculty of pharmacy approved the animal experiments, if they were carried out through in accordance with ethical standards and regulations with the ethics committee protocol approval number, p113-2021, which controls the care and use of laboratory animals.
 
Histopathological examinations
 
After the experimental, the ovary of each rat were collected and fixed in 10% neutral buffered formalin solution, cleared, dehydrated and embedded, tissue sections were cut at 5 μm and stained with hematoxylin and eosin for light microscopic (LM) examination (Dunn, 1974).
 
Biochemical assay
 
After 8 weeks of the experiment blood samples from each rat were collected directly from the heart for biochemical assay. The blood samples were spun at 3000 rpm for 10 min in a centrifuge. Serum Sample was aspirated with Pasteur pipettes into clean sample tubes. Hormonal levels assayed in female rats were follicle-stimulating hormone (FSH), progesterone and estrogen levels by using ELISA kits specific for rat (Pogrmic-Majkic et al., 2014).
 
Statistical analysis
 
Data were presented as mean± SE and compared by student’s t-test of means using the SPSS computer program.

RESULTS AND DISCUSSION

Normal histopathologic appearance was observed in the control group, Ovarian sections from the control group revealed primary, secondary and antral follicle-containing ovarian tissue was considered normal ovary structure. The ovary section of the treated group showed marked reduction in ovarian follicle numbers (F) and degeneration of interstitial cells and some follicles. Pre-ovulatory follicle (P), Degenerative oocytes (DO) with vacuolization in ooplasm with necrosis cells (N), Ovarian damage, follicular cell degeneration, vascular congestion (BV) and inflammation. The ovary section in the withdrawal control group was same with the control group with normal ovary showing well follicular development pre-ovulatory follicle (P) with a mature oocyte (O) surrounded by granulosa cells (C), normal histological arrangements of blood vessels (BV) and all layers are present. The Ovary section of withdrawal treated group, marked improvement in ovarian follicle numbers and healing of degeneration of interstitial cells and some follicles (F) (Fig 1-4). In the treated females, the levels of Progesterone hormone, significantly increased when compared with the control female group. In the withdrawal treated female group, the activities of the progesterone hormone were significantly increased when compared with the withdrawal control female group. In the treated females, the levels of estrogen hormone significantly (p<0.05) increased when compared with the control female group. In the withdrawal treated female group, the activities of the estrogen hormone were significantly increased (118.6±4.36) when compared with the withdrawal control female (109.8±3.77) group. In the treated female rats, the levels of FSH hormone, (p<0.05) significantly increased when compared with the control female group. In the withdrawal treated female group, the activities of the FSH hormone were significantly increased (11.5±1.44) when compared with the withdrawal control female group (9.5±0.28) (Fig 5-7).

Fig 1: Sections from the female rat ovary in control group showing primary oocyte (O), follicle (F). (H and E staining; magnification 400X).



Fig 2: Sections from the female rat ovary in treated group showing ovarian damage, follicular cell degeneration (F), vascular oedma (BV), necrosis cells (N) and extension of theca (Th). (H and E staining; magnification 1000X).



Fig 3: Sections from the rat ovary in withdrawal control group showing normal ovarian, pre-ovulatory follicle (P) with mature oocyte (O), granulosa cells (C). (H and E staining; magnification 400X).



Fig 4: Sections from the rat ovary in withdrawal treated group showed an increase in the number of follicles (F), mature oocyte (O), pre-ovulatory follicle (P), blood vessels (BV). (T.B staining; magnification 400X).



Fig 5: Effects of PPD on progesterone levels in the studied female groups for 8 weeks with a dose (10 mg/kg).



Fig 6: Effects of PPD on Estrogen levels in the studied female groups for 8 weeks with a dose (10 mg/kg).



Fig 7: Effects of PPD on FSH levels in the studied female groups for 8 weeks with a dose (10 mg/kg).


       
In the present study, changes were seen in treated females, the treated group, showed marker reduction in ovarian follicle numbers and degeneration of interstitial cells, degenerative oocytes with vacuolization in ooplasm with necrosis cells.
       
The reduction in ovarian follicle numbers and degeneration of interstitial cells agreed with the findings of Wang et al. (2022), who investigated the effect of PPD on ovary function in female rats. PPD can also impair the normal development and maturation of oocytes by damaging the spindle and chromosomal structure. Abnormal mitochondrial activity brought on by PPD exposure resulted in oocyte degeneration, apoptosis and elevated ROS levels.
       
The number of ovarian follicles in the treated group was substantially decreased and their level of FSH hormone were significantly higher. Consistent findings have been reported in the literature by Yu et al. (2021), who studied how the PPD metabolite N-monoacetyl-PPD (MAPPD) affected the development of rat blastocysts and ovarian function which indicates that while the number of antral follicles reduced, FSH increased and there was an obvious increase in the degree of oxidative stress. Cell regeneration and repair may be hampered by an excess of free radicals (Jain and Shakkarpude, 2024). Necrosis, vascular congestion, hemorrhage, leukocyte infiltration and edema were observed in ovarian and cardiac tissues due to oxidative stress (Delibas et al., 2018). A few investigations have shown that the ovary is vulnerable to the effects of lipid peroxidation because of damage brought on by hydroxyl free radicals (Lohiya et al., 2019).
       
Any component of the activity of hormones can be affected by an endocrine-disrupting material. Thus, some of the chemicals in hair dyes may have an impact on the endocrine system that controls sex hormones. It is reasonable to assume that circulating levels of sex hormones will serve as markers for sex hormone system disturbance (Nagata et al., 2009).
       
The present study showed that there was a significant increase in female hormone (FSH), estrogen and progesterone level after treatment with PPD for 8 weeks.
       
Progesterone and estrogen essential for the healthy operation of the female reproductive system. A normal, regular cycle was caused by the balance of the hormone interactions between progesterone and estrogens (Boubekri et al., 2009). Estrogen had several para/autocrine effects on the ovary, such as increasing ovarian weight, stimulating granule cells growth, increasing the action of FSH and reducing apoptosis by controlling the synthesis and release of gonadotropin from the hypothalamic-pituitary axis (Chou and Chen, 2018). Higher hair color application frequency was found to be marginally strongly associated with higher levels of estrogens, according to Nagata et al. (2015). However, several research on human and non-human animals indicated that variations in reproductive hormone levels are a factor in PPD (Bloch et al., 2000). Higher follicular phase FSH is associated with poor ovarian response (Appasamy et al., 2008).
       
The prevalent endocrine condition of Polycystic ovary syndrome (PCOS) was linked to abnormal estrogen and estrogen receptor (ER) function in female sex. Cellular processes include ovulation, cell cycle phase and cell proliferation, migration and invasion were all impacted by modifications in the signaling pathways associated with estrogen receptors (Xu et al., 2021). This was the association that our research developed, since the PPD impact caused an apparent increase in estrogen, which in effect caused ovarian cellular alterations. Quinine may have suppressed the progesterone and estrogen (Gbotolorun et al., 2018).

CONCLUSION

Oral treatment of paraphenylenediamine caused damage to the ovarian structure with changes in hormone levels. PPD might have a toxic effect on the ovaries and this requires further study and research.

ACKNOWLEDGEMENT

The present study was supported by Jeddah University, Department of Biology.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Amin, M.R., Khan, N.A., Ekram, R., Mallick, P.K., Khosru, O. and Islam, M.R. (2021). Hair dye can lead you dying’-Paraphenylenediamine poisoning with multi system dysfunction. Clin Case Rep J. 2: 1-3.

  2. Appasamy, M., Jauniaux, E., Serhal, P., Al-Qahtani, A., Groome, N.P. and Muttukrishna, S. (2008). Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones and response to gonadotropin stimulation. Fertility and Sterility. 89(4): 912-921.

  3. Bloch, M., Schmidt, P.J., Danaceau, M., Murphy, J., Nieman, L. and Rubinow, D.R. (2000). Effects of gonadal steroids in women with a history of postpartum depression. American Journal of Psychiatry. 157(6): 924-930.

  4. Boubekri, A., Gernigon-Spychalowicz, T., Khammar, F. and Exbrayat, J.M. (2009). Morphometry and immunohistochemistry of follicles growth and steroidogenesis in saharian wild sand rat, Psammomys obesus, ovary. Foliahistochemicaet  Cytobiologica. 47(5): 59-66.

  5. Brito, J.R.F.C.D. (2017). Evaluation of Hair Dyes Influence on the Epidermal Barrier using an in vitro Test (Doctoral dissertation).

  6. Cascão, M.R. (2021). Tattoos and Piercings and Health Consequences for Adolescents and Young Adults. Tattoos and Piercings and Health Consequences for Adolescents and Young Adults.

  7. Chou, C.H. and Chen, M.J. (2018). The effect of steroid hormones on ovarian follicle development. Vitamins and Hormones. 107: 155-175.

  8. de Groot, A.C. (2013). Side effects of henna and semi permanent ‘black henna’tattoos: A full review. Contact dermatitis. 69(1): 1-25.

  9. Delibas, I.B., Karaca, I., Yapca, O.E., Ingec, M., Kumbasar, S., Cimen, F.K. and Altuner, D. (2018). The effect of nizatidine on ovarian ischaemia/reperfusion injury in rats. Indian Journal of Animal Research. 52(10): 1488-1493. doi: 10.18805/ijar.B-934.

  10. Dunn, W.L. (1974). Handbook of Histopathological and Histochemical Techniques. Redwood: Bun, Ltd., Trowbridge and Esher.

  11. Eissa, U.B., Khogali, F., Mohamed, E.E., Osman, E.E., Elmahi, O.M. and Waggiallah, H.A. (2021). The potential effect of henna (Stone Dye) extract paraphenylenediamine on human blood cells, liver and renal function. Journal of Pharmaceutical Research International. pp. 192-200. 

  12. ElHelaly, H. and Shaker, S. (2014). Repeated dermal paraphenylenediamine exposure induces systemic biochemical and histological changes in adult male albino rats. Ain Shams Journal of Forensic Medicine and Clinical Toxicology. 23(2): 71-89.

  13. Elmanfe, G.M., Khreit, O.E., Abduljalil, O.A. and Abbas, N.M. (2022). Determination of para-phenylenediamine (PPD) in henna samples collected from libyan local markets using HPLC. Al-Mukhtar Journal of Sciences. 37(1): 13-21.

  14. Gbotolorun, S. C., Inikori, O., Bamisi, O.D., Osinubi, A.A.A. and Okanlawon, A.O. (2018). Quinine inhibits ovulation and produces oxidative stress in the ovary of cyclic Sprague- dawley rats. African Health Sciences. 18(2): 253-259.

  15. Hummdi, L.A. (2012). Histopathological alterations in renal tubules of female rats topically treated with paraphenylen diamine. World Applied Sciences Journal. 16(3): 376-388. 

  16. Ibrahim, S.Y. and Maguid, R.A. (2016). Evaluation of metals content among different cosmetic products in the Arabian market. J. Pharmacol. Clin. Toxicol. 4: 1062.

  17. Jain, A. and Shakkarpude, J. (2024). Oxidative stress: A biomarker for animal health and production: A review. Indian Journal of Animal Research. 58(1): 01-12. doi: 10.18805/IJAR.B-5300.

  18. Lohiya, A., Kumar, V. and Punia, J.S. (2019). Effect of imidacloprid on antioxidant status and histopathological changes in ovary and uterus of adult female wistar rats. Indian Journal of Animal Research. 53(8): 1014-1019. doi: 10.18805/ijar.B-3613.

  19. Malik, S., Qamar, K. and Rehman, S. (2016). Paraphenylene diamine induced histomorphological changes in rat ovary. Pakistan Journal of Pathology. 27(3): 124-129.

  20. Misirlioglu, E.D., Tanidir, M., Buyuktiryaki, B., Yuksek, S.K., Sahiner, N., guvenir, H. and Kocabas, C.N. (2022). Allergic contact dermatitis to temporary black henna tattoo due to sensitization to para-phenylenediamine. Asthma Allergy Immunology. 19(3): 183-185.

  21. Mohamed, O.R., Hassan, N.A., Mitwalli, I.S. and Hussein, A.Y. (2023). Toxicological effects of para-phenylenediamine on kidneys of female albino rats. Biannual Forensic Sciences and Toxicology Journal. 1(1): 1-12.

  22. Nagata, C., Nakamura, K., Oba, S., Hayashi, M., Takeda, N. and Yasuda, K. (2009). Association of intakes of fat, dietary fibre, soya isoflavones and alcohol with uterine fibroids in Japanese women. British Journal of Nutrition. 101(10):  1427-1431.

  23. Nagata, C., Wada, K., Tsuji, M., Hayashi, M., Takeda, N. and Yasuda, K. (2015). Association of hair dye use with circulating levels of sex hormones in premenopausal Japanese women. The European Journal of Public Health. 25(5): 895-899.

  24. Pogrmic-Majkic, K., Samardzija, D., Fa, S., Hrubik, J., Glisic, B., Kaisarevic, S. and Andric, N. (2014). Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: Evidence for premature luteinization. Biology of Reproduction.  91(5): 124-1.

  25. Venkatesan, G., Dancik, Y., Sinha, A., Kyaw, H. M., Srinivas, R., Dawson, Jr, T.L. and Pastorin, G. (2021). Development of novel alternative hair dyes to hazardous para- phenylenediamine. Journal of Hazardous Materials. 402: 123712.

  26. Venkatesan, G., Lim, Z.C., Karkhanis, A.V., Neupane, Y.R., Dancik, Y., Huang, C. and Pastorin, G. (2021). Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1). Archives of Toxicology. 96(1):153-165. doi: 10.1007/s00204-021- 03149-2.

  27. Waggas, A.M. (2011). Neuro and nephro-toxicity in rats topically treated with para-phenylene diamine. Journal of Toxicological Sciences. 3(3): 130-137.

  28. Wang, X., Zhao, X., Chen, Y., Wang, Q., Yang, H. and Xia, F. (2022). Para phenylenediamine deteriorates oocyte quality by impairing mitochondrial function. Environmental Toxicology. 37(7): 1803-1813.

  29. Xu, X.L., Deng, S.L., Lian, Z.X. and Yu, K. (2021). Estrogen receptors in polycystic ovary syndrome. Cells. 10(2): 459.

  30. Yu, L., Zhai, J., Wang, Y., Geng, Y., Chen, X., Wen, Y. and Liu, X. (2021). Exposure to N monoacetyl p phenylenediamine impaired ovarian function in mice. Journal of Applied Toxicology. 41(12): 2031-2041.
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    Review Article

    Histological and Physiological Studies on Adult Female Albino Rats Treated with Paraphenylenediamine

    M
    Mai Almosaibih1
    A
    Afrah ALrashidi1,*
    1Department of Biological Science, College of Science, University of Jeddah, Saudi Arabia.

    ABSTRACT

    Background: Women are exposed to Paraphenylenediamine (PPD) during hair dyeing in a variety of Arab nations, PPD may damage internal organs and result in death. The current study intends to assess the histological and physiological studies on female albino rats treated with Paraphenylenediamine.

    Methods: 24 females rats were used in the study and divided into four groups each with 6 rats: group1: control female group, rats will be treated, orally with distilled water for 8 weeks, group 2 treated female group, rats will receive 10 mg/kg B.W of PPD in 1 ml distilled water once daily for 8 weeks by oral gavage, group 3: withdrawal control female group, rats receive distilled water for 8 weeks and left untreated for two weeks after stopping treatment, group 4: withdrawal treated female group, rats will receive 10 mg/kg B.W of PPD for 8weeks and left untreated for two weeks after stopping treatment.

    Result: It was observed on microscopic examination that orally applied PPD solution affects histology of rat ovary marker reduction in ovarian follicle numbers and degeneration of interstitial cells and some follicles, with observable modifications in physiological processes in hormones, In the treated female group, the serum levels of progesterone, estrogen and FSH hormones significantly increased when compared with the control female group. It was concluded from the results that orally applied PPD solution induces histomorphology and physiological changes in the ovary of rats with 10 mg/kg dose.

    INTRODUCTION

    Paraphenylenediamine (PPD) is an aniline-related aromatic diamine that comes in the form of pinkish gray crystalline lumps with a high oxidation content (Eissa et al., 2021). It is employed as a photographic developing agent, an azo dye intermediate, an antioxidant and a rubber vulcanization accelerator, PPD is added to henna to speed up the dying and drying process, intensify and darken the color, improve the tattoo’s design pattern and make it last longer (Elhelaly and Shaker, 2014). Young adults and teenagers are gaining popular with temporary “black henna tattoos. (Cascão, 2021). PPD is present in most permanent hair dyes (Venkatesan et al., 2021), PPD can also be found as an antioxidant in rubber compounds as well as textile or fur dyes, photographic developing agents, dark-colored cosmetics, temporary tattoos, photocopying and printing inks, black rubber, oils, greases and gasoline (Elmanfe et al., 2022). it is also a component used in the production of polymers, aramid fibers, textile colors and dyes. No matter the brand, almost all natural, herbal, synthetic, or ammonia-free hair dyes include PPD (Brito, 2017).
           
    When applied topically or consumed, parapheny lenediamine can have local and systemic hazardous effects (Misirlioglu et al., 2022). Unfortunately, women are exposed to PPD during hair dying because PPD-containing hair dyes are far less expensive than other hair colors on the market (Mohamed et al., 2023). Hair dye has the most hazardous harmful consequences when it is absorbed through the skin, ingested, or inhaled. Depending on the dose and concentration of PPD, it might cause internal organ damage and death (Malik et al., 2016). Exposure to PPD occurs through skin, accidental ingestion, or inhalation of particles from hair dye formulation during dyeing. Acute exposure to high levels of Para phenylene diamine may cause severe dermatitis, asthma, renal failure, edema of face, neck and larynx. Cases of rhabdoms, acute tubular necrosis with acute renal failure and hepatic failure have been reported (Hummdi, 2012). Decrease in hemoglobin leading to anemia due to hemolytic effect of Paraphenylenediamine on red blood cells has been noticed in rats that received sub lethal doses of Paraphenylenediamine (Malik et al., 2016).
           
    Bandrowski’s base, an extremely poisonous molecule produced by PPD metabolism (Amin et al., 2021). When PPD is manufactured or used, workers may be exposed to it. Exposure can happen by inhalation, skin contact, eye contact and/or ingestion (Elmanfe et al., 2022). So, the current study aims to evaluate the effect of PPD on female reproductive system.
           
    In 1924, a hairdresser reported the first incidence of PPD toxicity due to exposure from PPD dye processing (Waggas, 2011). Since it has been discovered that cosmetics can have both local and systemic effects, they have attracted a lot of attention (de Groot, 2013). Contrary to popular perception, henna tattoos can cause allergic responses, some of which can be quite severe. PPD is one of the substances used in henna tattoos. PPD, a severe contact sensitizer, can be found in black henna tattoos and hair dyes (Misirlioglu et al., 2022). In oxidative hair dyes, paraphenylenediamine (PPD) is one of the most common compounds utilized. However, its use has been linked to negative health impacts (Venkatesan et al., 2021).
        
    Due to research showing that cosmetic substances possess both local and systemic effects, there was much of interest in these substances in the past several years. PPD speeds up the coloring process when used in hair dyes (Ibrahim and Maguid, 2016). There is ample evidence of paraphenylenediamine’s effects on the liver, pancreas, kidneys and heart; however, the effects on the female reproductive system are less established. This study was designed to investigate the histological and physiological effects of paraphenylenediamine on the ovary of rats.

    MATERIALS AND METHODS

    Chemicals
     
    The teste chemical paraphenylenediamine (PPD), CAS No (106-50-3) was purchased from Sigma Chemical Company.
     
    Animals experimental design
     
    A total of 24 adult female rats, (body weight 160±200 g), (n=6) was used in this study. the rat was from the experimental animal unit of faculty of pharmacy. King Abdul-Aziz University, Jeddah, Saudi Arabia (2022). Rats were adjusted to the laboratory conditions for one week before the activation of the experiments. The animals were housed in standard spotless plastic cages and kept in controlled laboratory status of temperature (20±1°C), humidity (65%) and 12 h light: dark cycle. The animals had free line tap water and are ad libitum on normal commercial chow diet.
     
    The rats were divided into four groups (n=6):
     
    Control group
     
    Female rats were treated orally with distilled water.
     
    Treated group
     
    Experimental group rats were administered 10 mg/kg B.W of PPD in 1 ml distilled water once daily for 8 weeks by oral gavage.
     
    Withdrawal control group
     
    Female rats of control group fed distilled water for 8 weeks and left untreated for two weeks after stopping treatment.
     
    Withdrawal treated group
     
    Female rats fed 10 mg/kg B.W of PPD for 8weeks were left untreated for two weeks after stopping treatment.
     
    Ethics approval for animal experimentation
     
    The King Abdul-Aziz University faculty of pharmacy approved the animal experiments, if they were carried out through in accordance with ethical standards and regulations with the ethics committee protocol approval number, p113-2021, which controls the care and use of laboratory animals.
     
    Histopathological examinations
     
    After the experimental, the ovary of each rat were collected and fixed in 10% neutral buffered formalin solution, cleared, dehydrated and embedded, tissue sections were cut at 5 μm and stained with hematoxylin and eosin for light microscopic (LM) examination (Dunn, 1974).
     
    Biochemical assay
     
    After 8 weeks of the experiment blood samples from each rat were collected directly from the heart for biochemical assay. The blood samples were spun at 3000 rpm for 10 min in a centrifuge. Serum Sample was aspirated with Pasteur pipettes into clean sample tubes. Hormonal levels assayed in female rats were follicle-stimulating hormone (FSH), progesterone and estrogen levels by using ELISA kits specific for rat (Pogrmic-Majkic et al., 2014).
     
    Statistical analysis
     
    Data were presented as mean± SE and compared by student’s t-test of means using the SPSS computer program.

    RESULTS AND DISCUSSION

    Normal histopathologic appearance was observed in the control group, Ovarian sections from the control group revealed primary, secondary and antral follicle-containing ovarian tissue was considered normal ovary structure. The ovary section of the treated group showed marked reduction in ovarian follicle numbers (F) and degeneration of interstitial cells and some follicles. Pre-ovulatory follicle (P), Degenerative oocytes (DO) with vacuolization in ooplasm with necrosis cells (N), Ovarian damage, follicular cell degeneration, vascular congestion (BV) and inflammation. The ovary section in the withdrawal control group was same with the control group with normal ovary showing well follicular development pre-ovulatory follicle (P) with a mature oocyte (O) surrounded by granulosa cells (C), normal histological arrangements of blood vessels (BV) and all layers are present. The Ovary section of withdrawal treated group, marked improvement in ovarian follicle numbers and healing of degeneration of interstitial cells and some follicles (F) (Fig 1-4). In the treated females, the levels of Progesterone hormone, significantly increased when compared with the control female group. In the withdrawal treated female group, the activities of the progesterone hormone were significantly increased when compared with the withdrawal control female group. In the treated females, the levels of estrogen hormone significantly (p<0.05) increased when compared with the control female group. In the withdrawal treated female group, the activities of the estrogen hormone were significantly increased (118.6±4.36) when compared with the withdrawal control female (109.8±3.77) group. In the treated female rats, the levels of FSH hormone, (p<0.05) significantly increased when compared with the control female group. In the withdrawal treated female group, the activities of the FSH hormone were significantly increased (11.5±1.44) when compared with the withdrawal control female group (9.5±0.28) (Fig 5-7).

    Fig 1: Sections from the female rat ovary in control group showing primary oocyte (O), follicle (F). (H and E staining; magnification 400X).



    Fig 2: Sections from the female rat ovary in treated group showing ovarian damage, follicular cell degeneration (F), vascular oedma (BV), necrosis cells (N) and extension of theca (Th). (H and E staining; magnification 1000X).



    Fig 3: Sections from the rat ovary in withdrawal control group showing normal ovarian, pre-ovulatory follicle (P) with mature oocyte (O), granulosa cells (C). (H and E staining; magnification 400X).



    Fig 4: Sections from the rat ovary in withdrawal treated group showed an increase in the number of follicles (F), mature oocyte (O), pre-ovulatory follicle (P), blood vessels (BV). (T.B staining; magnification 400X).



    Fig 5: Effects of PPD on progesterone levels in the studied female groups for 8 weeks with a dose (10 mg/kg).



    Fig 6: Effects of PPD on Estrogen levels in the studied female groups for 8 weeks with a dose (10 mg/kg).



    Fig 7: Effects of PPD on FSH levels in the studied female groups for 8 weeks with a dose (10 mg/kg).


           
    In the present study, changes were seen in treated females, the treated group, showed marker reduction in ovarian follicle numbers and degeneration of interstitial cells, degenerative oocytes with vacuolization in ooplasm with necrosis cells.
           
    The reduction in ovarian follicle numbers and degeneration of interstitial cells agreed with the findings of Wang et al. (2022), who investigated the effect of PPD on ovary function in female rats. PPD can also impair the normal development and maturation of oocytes by damaging the spindle and chromosomal structure. Abnormal mitochondrial activity brought on by PPD exposure resulted in oocyte degeneration, apoptosis and elevated ROS levels.
           
    The number of ovarian follicles in the treated group was substantially decreased and their level of FSH hormone were significantly higher. Consistent findings have been reported in the literature by Yu et al. (2021), who studied how the PPD metabolite N-monoacetyl-PPD (MAPPD) affected the development of rat blastocysts and ovarian function which indicates that while the number of antral follicles reduced, FSH increased and there was an obvious increase in the degree of oxidative stress. Cell regeneration and repair may be hampered by an excess of free radicals (Jain and Shakkarpude, 2024). Necrosis, vascular congestion, hemorrhage, leukocyte infiltration and edema were observed in ovarian and cardiac tissues due to oxidative stress (Delibas et al., 2018). A few investigations have shown that the ovary is vulnerable to the effects of lipid peroxidation because of damage brought on by hydroxyl free radicals (Lohiya et al., 2019).
           
    Any component of the activity of hormones can be affected by an endocrine-disrupting material. Thus, some of the chemicals in hair dyes may have an impact on the endocrine system that controls sex hormones. It is reasonable to assume that circulating levels of sex hormones will serve as markers for sex hormone system disturbance (Nagata et al., 2009).
           
    The present study showed that there was a significant increase in female hormone (FSH), estrogen and progesterone level after treatment with PPD for 8 weeks.
           
    Progesterone and estrogen essential for the healthy operation of the female reproductive system. A normal, regular cycle was caused by the balance of the hormone interactions between progesterone and estrogens (Boubekri et al., 2009). Estrogen had several para/autocrine effects on the ovary, such as increasing ovarian weight, stimulating granule cells growth, increasing the action of FSH and reducing apoptosis by controlling the synthesis and release of gonadotropin from the hypothalamic-pituitary axis (Chou and Chen, 2018). Higher hair color application frequency was found to be marginally strongly associated with higher levels of estrogens, according to Nagata et al. (2015). However, several research on human and non-human animals indicated that variations in reproductive hormone levels are a factor in PPD (Bloch et al., 2000). Higher follicular phase FSH is associated with poor ovarian response (Appasamy et al., 2008).
           
    The prevalent endocrine condition of Polycystic ovary syndrome (PCOS) was linked to abnormal estrogen and estrogen receptor (ER) function in female sex. Cellular processes include ovulation, cell cycle phase and cell proliferation, migration and invasion were all impacted by modifications in the signaling pathways associated with estrogen receptors (Xu et al., 2021). This was the association that our research developed, since the PPD impact caused an apparent increase in estrogen, which in effect caused ovarian cellular alterations. Quinine may have suppressed the progesterone and estrogen (Gbotolorun et al., 2018).

    CONCLUSION

    Oral treatment of paraphenylenediamine caused damage to the ovarian structure with changes in hormone levels. PPD might have a toxic effect on the ovaries and this requires further study and research.

    ACKNOWLEDGEMENT

    The present study was supported by Jeddah University, Department of Biology.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Amin, M.R., Khan, N.A., Ekram, R., Mallick, P.K., Khosru, O. and Islam, M.R. (2021). Hair dye can lead you dying’-Paraphenylenediamine poisoning with multi system dysfunction. Clin Case Rep J. 2: 1-3.

    2. Appasamy, M., Jauniaux, E., Serhal, P., Al-Qahtani, A., Groome, N.P. and Muttukrishna, S. (2008). Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones and response to gonadotropin stimulation. Fertility and Sterility. 89(4): 912-921.

    3. Bloch, M., Schmidt, P.J., Danaceau, M., Murphy, J., Nieman, L. and Rubinow, D.R. (2000). Effects of gonadal steroids in women with a history of postpartum depression. American Journal of Psychiatry. 157(6): 924-930.

    4. Boubekri, A., Gernigon-Spychalowicz, T., Khammar, F. and Exbrayat, J.M. (2009). Morphometry and immunohistochemistry of follicles growth and steroidogenesis in saharian wild sand rat, Psammomys obesus, ovary. Foliahistochemicaet  Cytobiologica. 47(5): 59-66.

    5. Brito, J.R.F.C.D. (2017). Evaluation of Hair Dyes Influence on the Epidermal Barrier using an in vitro Test (Doctoral dissertation).

    6. Cascão, M.R. (2021). Tattoos and Piercings and Health Consequences for Adolescents and Young Adults. Tattoos and Piercings and Health Consequences for Adolescents and Young Adults.

    7. Chou, C.H. and Chen, M.J. (2018). The effect of steroid hormones on ovarian follicle development. Vitamins and Hormones. 107: 155-175.

    8. de Groot, A.C. (2013). Side effects of henna and semi permanent ‘black henna’tattoos: A full review. Contact dermatitis. 69(1): 1-25.

    9. Delibas, I.B., Karaca, I., Yapca, O.E., Ingec, M., Kumbasar, S., Cimen, F.K. and Altuner, D. (2018). The effect of nizatidine on ovarian ischaemia/reperfusion injury in rats. Indian Journal of Animal Research. 52(10): 1488-1493. doi: 10.18805/ijar.B-934.

    10. Dunn, W.L. (1974). Handbook of Histopathological and Histochemical Techniques. Redwood: Bun, Ltd., Trowbridge and Esher.

    11. Eissa, U.B., Khogali, F., Mohamed, E.E., Osman, E.E., Elmahi, O.M. and Waggiallah, H.A. (2021). The potential effect of henna (Stone Dye) extract paraphenylenediamine on human blood cells, liver and renal function. Journal of Pharmaceutical Research International. pp. 192-200. 

    12. ElHelaly, H. and Shaker, S. (2014). Repeated dermal paraphenylenediamine exposure induces systemic biochemical and histological changes in adult male albino rats. Ain Shams Journal of Forensic Medicine and Clinical Toxicology. 23(2): 71-89.

    13. Elmanfe, G.M., Khreit, O.E., Abduljalil, O.A. and Abbas, N.M. (2022). Determination of para-phenylenediamine (PPD) in henna samples collected from libyan local markets using HPLC. Al-Mukhtar Journal of Sciences. 37(1): 13-21.

    14. Gbotolorun, S. C., Inikori, O., Bamisi, O.D., Osinubi, A.A.A. and Okanlawon, A.O. (2018). Quinine inhibits ovulation and produces oxidative stress in the ovary of cyclic Sprague- dawley rats. African Health Sciences. 18(2): 253-259.

    15. Hummdi, L.A. (2012). Histopathological alterations in renal tubules of female rats topically treated with paraphenylen diamine. World Applied Sciences Journal. 16(3): 376-388. 

    16. Ibrahim, S.Y. and Maguid, R.A. (2016). Evaluation of metals content among different cosmetic products in the Arabian market. J. Pharmacol. Clin. Toxicol. 4: 1062.

    17. Jain, A. and Shakkarpude, J. (2024). Oxidative stress: A biomarker for animal health and production: A review. Indian Journal of Animal Research. 58(1): 01-12. doi: 10.18805/IJAR.B-5300.

    18. Lohiya, A., Kumar, V. and Punia, J.S. (2019). Effect of imidacloprid on antioxidant status and histopathological changes in ovary and uterus of adult female wistar rats. Indian Journal of Animal Research. 53(8): 1014-1019. doi: 10.18805/ijar.B-3613.

    19. Malik, S., Qamar, K. and Rehman, S. (2016). Paraphenylene diamine induced histomorphological changes in rat ovary. Pakistan Journal of Pathology. 27(3): 124-129.

    20. Misirlioglu, E.D., Tanidir, M., Buyuktiryaki, B., Yuksek, S.K., Sahiner, N., guvenir, H. and Kocabas, C.N. (2022). Allergic contact dermatitis to temporary black henna tattoo due to sensitization to para-phenylenediamine. Asthma Allergy Immunology. 19(3): 183-185.

    21. Mohamed, O.R., Hassan, N.A., Mitwalli, I.S. and Hussein, A.Y. (2023). Toxicological effects of para-phenylenediamine on kidneys of female albino rats. Biannual Forensic Sciences and Toxicology Journal. 1(1): 1-12.

    22. Nagata, C., Nakamura, K., Oba, S., Hayashi, M., Takeda, N. and Yasuda, K. (2009). Association of intakes of fat, dietary fibre, soya isoflavones and alcohol with uterine fibroids in Japanese women. British Journal of Nutrition. 101(10):  1427-1431.

    23. Nagata, C., Wada, K., Tsuji, M., Hayashi, M., Takeda, N. and Yasuda, K. (2015). Association of hair dye use with circulating levels of sex hormones in premenopausal Japanese women. The European Journal of Public Health. 25(5): 895-899.

    24. Pogrmic-Majkic, K., Samardzija, D., Fa, S., Hrubik, J., Glisic, B., Kaisarevic, S. and Andric, N. (2014). Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: Evidence for premature luteinization. Biology of Reproduction.  91(5): 124-1.

    25. Venkatesan, G., Dancik, Y., Sinha, A., Kyaw, H. M., Srinivas, R., Dawson, Jr, T.L. and Pastorin, G. (2021). Development of novel alternative hair dyes to hazardous para- phenylenediamine. Journal of Hazardous Materials. 402: 123712.

    26. Venkatesan, G., Lim, Z.C., Karkhanis, A.V., Neupane, Y.R., Dancik, Y., Huang, C. and Pastorin, G. (2021). Investigations on detoxification mechanisms of novel para-phenylenediamine analogues through N-acetyltransferase 1 (NAT-1). Archives of Toxicology. 96(1):153-165. doi: 10.1007/s00204-021- 03149-2.

    27. Waggas, A.M. (2011). Neuro and nephro-toxicity in rats topically treated with para-phenylene diamine. Journal of Toxicological Sciences. 3(3): 130-137.

    28. Wang, X., Zhao, X., Chen, Y., Wang, Q., Yang, H. and Xia, F. (2022). Para phenylenediamine deteriorates oocyte quality by impairing mitochondrial function. Environmental Toxicology. 37(7): 1803-1813.

    29. Xu, X.L., Deng, S.L., Lian, Z.X. and Yu, K. (2021). Estrogen receptors in polycystic ovary syndrome. Cells. 10(2): 459.

    30. Yu, L., Zhai, J., Wang, Y., Geng, Y., Chen, X., Wen, Y. and Liu, X. (2021). Exposure to N monoacetyl p phenylenediamine impaired ovarian function in mice. Journal of Applied Toxicology. 41(12): 2031-2041.
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