Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Histone deacetylase inhibition leading to testicular toxicity||AdverseOutcome|
|Adult, reproductively mature||Moderate|
Key Event Description
It is hypothesized that the testicular effects of 1,6-dimethoxyhexane (DMH) are caused by its metabolism to MAA [Wade, 2006, Poon, 2004]. MAA produces testicular and thymic atrophy [Miller, 1982, Moss, 1985]. The spermatogenic stages in which the toxicity of MAA is induced are on the patchytene spermatocytes immediately before and during meiotic division, which are Stages XII-XIV of the cycle in the rat and the early pachytene spermatocytes at stages I-IV of the cycle. Dead germ cells can be seen as soon as 12 hours after the treatment of MAA [Casarett & Doull’s 7th edition].
How It Is Measured or Detected
The weights of testes of MAA-treated rats were measured to detect the testicular atrophy [Foster, 1983]. Since zinc concentration has been shown to play an important role in the production of testicular injury by compounds, the effects of EGME and MAA on urinary zinc excretion and testicular zinc content was examined [Foster, 1983]. Testis were fixed for observations for light microscopy or transmission electron microscopy [McDowell, 1976, Mercantepe, 2018]. The testicular tissue structure was observed whether there are normal germinal epithelial cells and Leydig cells [Mercantepe, 2018]. Changes in sperm were measured by computer-assisted sperm analysis [Foote, 1995]. For the testis cell analysis, fresh testes were dispersed using a two-stage enzymatic digestion and incubated in BSA containing collagenase and DNase I [Wade, 2006]. The seminiferous tubules were further digested and cells were fixed in ice-cold 70% ethanol [Wade, 2006]. Relative proportions of spermatogenic cell populations were assessed in fixed cells using a flow cytometeric method [Wade, 2006]. The principle of the test is that spermatogenic cells, as they differentiate from normal diploid spermatogonial stem cells through to mature spermatozoa with a highly condensed haploid complement of DNA, progress through various intermediate stages with differing nuclear DNA content and cellular content of mitochondria. Relative proportions of cells in each population were calculated with WinList software [Wade, 2006]. For the assessment of sperm morphology, eosin-stained sperm collected from the cauda epididymis were smeared onto two glass slides per sample, air-dried, and cover-slipped. At least 200 sperm on each slide were examined for the proportion of sperm with abnormal head (overhooked, blunt hook, banana-shaped, amorphous, or extremely oversized) or tail (twisted, bent, corkscrew, double. multiple) by one individual unaware of animal number or treatment [Wade, 2006]. For the measurement of the total number of condensed spermatids per testis, a weighed portion of the parenchyma from the left testis, as representative of the whole organ as possible, was homogenized [Wade, 2006]. For the measurement of the total number of sperm in the cauda epididymis, whole cauda and associated sperm suspension in DPBS were thawed on ice and homogenized [Wade, 2006]. Sperm or homogenization-resistant spermatid nuclei densities were calculated from the average number of nuclei and were expressed as total or as per gram of epididymis or testis weight [Wade, 2006]. For the determination of total LDH and LDH-X in supernatant of the homogenized testis fragment, enzyme activity was measured by monitoring extinction of NAD absorbance [Wade, 2006].
Domain of Applicability
There are some evidences on testicular toxicity induced by HDAC inhibitors.
・EGME or MAA treatment induced the testicular damage in rat (Rattus norvegicus) [Foster, 1983].
・EGME were shown to deplete the spermatocytes in CD-1 mice (Mus musculus) and CD rats (Rattus norvegicus), principally pachytene cells, but with other stages affected with increasing dose [Anderson, 1987].
・The testicular lesions induced by 2-methoxyethanol were observed in rats (Rattus norvegicus) and guinea pigs (Cavia porcellus), which are different in onset, characteristics and severity [Ku, 1984].
・EGME has effects in disruption of spermatogenesis in rabbits (Oryctolagus cuniculus) [Foote, 1995].
・Dimethoxyhexane (DMH) induces testicular toxicity such as spermatocyte death in seminiferous tubule stages I-IV and stages XII-XIV and MAA increase in urine in Sprague-Dawley rats (Rattus norvegicus).
Regulatory Significance of the Adverse Outcome
The testicular toxicity assessment is important for assessing the side effects of the medicines such as anti-cancer drugs. The unexpected effects may be predicted with this AO.
Wade MG et al. (2006) Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers. Toxicol Sci 89:304-313
Poon R et al. (2004) Short-term oral toxicity of pentyl ether, 1,4-diethoxybutane, and 1,6-dimethoxyhexane in male rats. Toxicol Sci 77:142-150
Miller R et al. (1982) Toxicity of methoxyacetic acid in rats. Fundam Appl Toxicol 2:158-160
Moss EJ et al. (1985) The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol Appl Pharmacol 79:480-489
Casarett & Doull’s Toxicology, the Basic Science of Poisons, 7th Edition, Edited by Curtis D. Klaassen, Chapter 20 Toxic responses of the reproductive system
Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rats. Toxicol Appl Pharmacol 69:385-399
McDowell EM and Trump BF. (1976) Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414
Mercantepe T et al. (2018) Protective effects of amifostine, curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats. Exp Ther Med 15:3404-3412
Foote RH et al. (1995) Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits. Reprod Toxicol 9:527-539
Anderson D et al. (1987) Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males. Teratog Carcinog Mutagen 7:141-158
Ku WW et al. (1994) Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs. Exp Mol Pathol 61:119-133