Giemsa-stained pseudo-micronuclei in rat skin treated with vitamin D3 analog, pefcalcitol

Abstract

BackgroundPefcalcitol, an analog of vitamin D3 (VD3), is an anti-psoriatic drug candidate that is designed to achieve much higher pharmacological effects, such as keratinocyte differentiation, than those of VD3, with fewer side effects. Genotoxicity of the compound was evaluated in a rat skin micronucleus (MN) test.ResultsIn the rat skin MN test, pefcalcitol showed positive when specimens were stained with Giemsa, whereas neither an in vitro chromosome aberration test in CHL cells nor an in vivo bone marrow MN test in rats indicated clastogenicity. To elucidate the causes of the discrepancy, the MN specimens were re-stained with acridine orange (AO), a fluorescent dye specific to nucleic acid, and the in vivo clastogenicity of the compound in rat skin was re-evaluated. The MN-like granules that had been stained by Giemsa were not stained by AO, and AO-stained specimens indicated that pefcalcitol did not increase the frequency of micronucleated (MNed) cells. Histopathological evaluation suggested that the MN-like granules in the epidermis were keratohyalin granules contained in keratinocytes, which had highly proliferated after treatment with pefcalcitol.ConclusionsPefcalcitol was concluded to be negative in the rat skin MN test. The present study demonstrated that Giemsa staining gave a misleading positive result in the skin MN test, because Giemsa stained keratohyalin granules.

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2‐mercaptobenzothiazole generates γ‐H2AX via CYP2E1‐dependent production of reactive oxygen species in urothelial cells

“…In recent years, phosphorylation of histone H2AX, a rare component of histone H2A, has become a powerful tool for detecting a variety of genotoxic events. [ 10–13 ] Although γ‐H2AX was originally identified as an early event after the direct formation of DNA double‐strand breaks (DSBs) after exposure to ionizing radiation, [ 14 ] it is now known that even if the primary form of DNA damage is one other than DSBs (such as single‐strand breaks, DNA adducts, oxidized bases, and cross‐links), DSBs are indirectly generated and γ‐H2AX is induced by the collision stress of replication forks at the damage site or by damage repair. [ 10 ] γ‐H2AX is useful for the detection of multiple types of DNA damage and is considerably more sensitive than other available techniques such as pulsed‐field gel electrophoresis and comet assays for the detection of DNA damage.…”