Objectives: Investigation of osteoblastic responses to oxidative stress, induced by C-reactive protein (CRP) and
IL-6 and ameliorating effects of doxycycline (Dox); using assays for 5-alpha dihydrotestosterone (DHT) as an antioxidant
marker of healing. IL-6 and CRP are risk markers of periodontitis and prevalent comorbidities in periodontitis subjects.
Methods: Confluent monolayer cultures of osteoblasts were incubated with radiolabelled testosterone (14C-T) as substrate,
in the presence or absence (Control) of pre-determined optimal concentrations of CRP, IL-6, Dox; alone and in
combination (n=8) for 24h in MEM. The eluent was solvent-extracted for steroid metabolites. They were separated using
TLC in a benzene/ acetone solvent system 4:1 v/v; and quantified using radioisotope scanning. The identity of formed metabolites
was confirmed using the mobility of cold standards added to the samples and disclosed in iodine. Further confirmation
of the authenticity of DHT was carried out by combined gas chromatrography-mass spectrometry, after derivatization
to pentafluorobenzyloxime trimethyl silyl ether. Results: The yields of DHT from 14C-testosterone showed 2-fold
and 1.8-fold- inhibition in response to IL-6 and CRP respectively and 28% stimulation in response to Dox, via the 5-alpha
reductase pathway. The combination of IL-6 + CRP showed a 2-fold reduction in the yields of DHT, elevated to control
values when combined with Dox (n=8; p<0.001). Yields of 4-androstenedione showed an inverse relationship to those of
DHT, in response to the agents tested, in keeping with the 17-beta hydroxysteroid dehydrogenase pathway. Conclusions:
Inhibition of DHT synthesis in osteoblasts by IL-6 and CRP was overcome by doxycycline. Oxidative actions of IL-6 and
CRP; and antioxidant actions of Dox are reinforced by the metabolic yields of DHT in response to agents tested. Using a
novel metabolically active model allows closer extrapolation to in vivo conditions; in the context of adjunctive therapeutic
applications for periodontitis and prevalent comorbidities.
Keywords: Antioxidant responses, CRP, doxycycline, IL-6, DHT and AR, osteoblasts, periodontitis and systemic comorbidities,
redox healing, risk markers.
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