Testicular toxicity following chronic codeine administration is via oxidative DNA damage and up-regulation of NO/TNF-α and caspase 3 activities

Abstract

Background: Codeine, a 3-methylmorphine, and other related opioids have been implicated in androgen suppression, although the associated mechanisms remain unclear.

Aim: Therefore, the objective of the current study was to elucidate the in vivo molecular mechanisms underlying codeine-induced androgen suppression.

Methods: This study made use of twenty-one healthy male rabbits, distributed into three groups randomly, control and codeine-treated groups. The control had 1ml of normal saline daily p.o. The codeine-treated groups received either 4mg/kg b.w of codeine or 10mg/kg b.w of codeine p.o. for six weeks. Reproductive hormonal profile, testicular weight, testicular enzymes, oxidative and inflammatory parameters, testicular DNA fragmentation, histological examination and apoptosis marker were evaluated to examine the effects of codeine use.

Key findings: Oral administration of codeine resulted in testicular atrophy and alterations in testicular histomorphology, elevated testicular enzymes, and suppression of circulatory and intra-testicular testosterone. These changes were associated with a marked rise in oxidative markers and decline in the activities of testicular enzymatic antioxidants, as well as oxidative DNA damage, inflammatory response, testicular DNA fragmentation, and caspase-dependent apoptosis (p<0.05).

Significance: In conclusion, chronic codeine use resulted in testicular degeneration and testosterone suppression, which is attributable to TNF-α/nitric oxide-/oxidative stress-mediated caspase-dependent apoptotic testicular cell death and loss of testicular function.

Fig 1. Effect of chronic codeine use on testicular enzymes activities.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 2. Effect of chronic codeine use on testicular total protein and glucose.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 3. Effect of chronic codeine use on male reproductive hormones.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 4. Effect of chronic codeine use on hormonal fertility indices.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 5. Effect of chronic codeine use on testicular oxidative stress markers.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 6. Effect of chronic codeine use on the activities of testicular enzymatic anti-oxidants.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 7. Effect of chronic codeine use on testicular oxidative DNA damage.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 8. Effect of chronic codeine use on some inflammatory markers.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 9. Effect of chronic codeine use on testicular DNA fragmentation.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 10. Effect of chronic codeine use on testicular caspase 3 activity.

*p < 0.05 vs control, #p < 0.05 vs low-dose codeine.

Fig 11. Photomicrographs of testicular sections stained by Haematoxylin and Eosin.

The control shows some normal seminiferous tubules containing normal germ cells including spermatogonia cells and sertoli cells, and normal maturation stages with presence of spermatozoa within their lumen(white arrow). There very few tubules show maturation arrest at secondary stage of maturation (black arrow). The interstitial spaces show normal leydig cells (slender arrow). The rabbits on low-dose codeine show very poor architecture. The seminiferous tubules show double cell layer or thickened propria indicative of cessation of spermatogenesis. They exhibit vacuolation, sloughed germ cells and maturation arrest and show giant cells (black arrow). There is evidence of vascular congestion (green arrow). The interstitial spaces appear normal (slender arrow). The animals on high-dose codeine show very poor architecture. The seminiferous tubules show double cell layer or thickened propria indicative of cessation of spermatogenesis. They exhibit vacuolation, sloughed germ cells and maturation arrest and show giant cells (black arrow). The interstitial spaces appear normal (slender arrow).

Fig 12. Graphical abstract showing the proposed mechanism of codeine-induced testicular toxicity.