Acute Assessment of Azorubine Exposure on Testosterone, Progesterone, Estradiol, Follicle Stimulating Hormone, Luteinizing Hormone, and Prolactin in Male and Female Albino Rats

Abstract

A study of azorubine toxicity on reproductive hormones and gonads of albino rats was carried out. A total of 80 albino rats weighing approximately 0.15kg were used, of which a total of 40 rats were used for the intraperitoneal study and another 40 were used for the oral study. The rats were divided randomly into 10 groups of 4 rats each for both the intraperitoneal and oral treatments. The groups were designed A, B, C, D, E, F, G, H, I, and J for the pilot study to determine the LD100 while the acute research only considered the groups A, B, C, D, E, and F following the determination of the LD100.The rats were treated with varying doses of azorubine for 48 hours and blood samples were collected using cardiac puncture. Hormonal parameters like testosterone, estradiol, progesterone, follicle stimulating hormone, luteinizing hormone, and prolactin were analysed from blood samples collected from the treated rats using the ELISA method. The H & E staining technique was used for histologically evaluating the ovaries and testes. Statistical analysis was done using GraphPad Prism, version 9.02, and results obtained were expressed as Mean±SD. In terms of pilotstudy, doses used for the intraperitoneal administration were 0.0g/kg, 0.17g/kg, 0.50g/kg, 1.0g/kg, 1.53g/kg, 2.0g/kg, 2.5g/kg, 3.33g/kg, 4.17g/kg, and 5.0g/kg while doses used orally were 0.0g/kg, 5.0g/kg, 10.0g/kg, 12.5g/kg, 17.5g/kg, 22.5g/kg, 25.0g/kg, 32.5g/kg, 37.5g/kg, and 40.0g/kg. In terms of acute study, the intraperitoneally treated groups were designated ACIP, BCIP, CCIP, DCIP, ECIP, and FCIP with doses of 0.0g/kg, 0.17g/kg, 0.50g/kg, 1.0g/kg, 1.53g/kg and 2.0g/kg respectively. In contrast, that of the oral were designated ACO, BCO, CCO, DCO, ECO, and FCO were given doses of 0.0g/kg, 5.0g/kg, 10.0g/kg, 12.5g/kg, 17.5g/kg, and 22.5g/kg respectively after LD100 determination. The results of the acute study showed significant dose-dependent decreases in testosterone in male rats while in the female rats, significantly reduced values of follicle stimulating hormone were observed. However, luteinizing hormone, progesterone, estrogen, and prolactin indicated significantly higher values in rats treated with high doses of azorubine. In addition, estrogen, progesterone, and luteinizing hormones indicated dose-dependent increased values in the azorubine treated rats. Non-significant decreases were observed in the absolute weight of the testes and ovaries. Histological evaluation of the testes indicated vacuolated portions of spermatogonia layer, pycnosis, scanty, and distorted Leydig cells, distorted flagella, and basement membrane at a dose of 22.5g/kg given orally. The ovaries showed intense lutein cells with yellowish colouration, follicular cells, and antrum of ovarian follicle surrounded by granulosa cells with vacuolations. Conclusively, azorubine could be described as an endocrine disruptor and a precocious agent especially when consumed in high doses even for a very short period of 48 hours as indicated by increased presence of lutein cells, Graafian follicles, estrogen, progesterone, prolactin, and luteinizing hormone in female rats. Spermatogenesis could also be impeded in male rats due to an induced significant fall in testosterone concentration alongside testicular damage. Therefore, azorubine should be used with caution and high doses should be avoided.