Table 1.

Antioxidant agents in GH and GO: laboratory studies

Reference	Fibroblast source	Compound	Main finding
Lisi et al. [59]	Orbital tissue from 5 GO patients Normal orbital tissue from 5 patients who underwent eye surgery for unrelated reasons	Quercetin Cells were incubated with quercetin or, as controls with quercitrin or rutin Cell proliferation, cell necrosis, apoptosis, and HA were measured	Reduction of cell proliferation and HA release in GO fibroblasts: quercetin, but not rutin or quercitrin, reduced cell proliferation, with no difference between GO and control fibroblasts; the effect of quercetin on proliferation was due to necrosis and cell cycle blockade, whereas apoptosis was unaffected; quercetin reduced HA in the cell media, with no difference between GO and control fibroblasts
Tsai et al. [46]	Orbital tissue from 7 GO patients Normal orbital tissue from 5 patients who received surgery for noninflammatory conditions	N-acetylcysteine or vitamin C Cells were treated with various H2O2 concentrations or pretreated with N-acetylcysteine or vitamin C, followed by treatment with H2O2 Cell proliferation, cell necrosis, apoptosis, and HA production were measured	Reduction of proliferation and release of cytokines in GO fibroblasts: when GO fibroblasts were exposed to H2O2 at a concentration of 50 µM or above, cytotoxicity was observed; lower concentrations of H2O2 (3.125–25 µM) increased the survival of GO fibroblasts; this biphasic effect was not found in control fibroblasts; 6.25 µM H2O2 led to an increase in TGF-β1, IL-1β, and superoxide anion in GO fibroblasts but not in control fibroblasts; pretreatment with N-acetylcysteine or vitamin C reversed the enhanced proliferation and the production of TGF-β1, IL-1β, and superoxide anion of GO fibroblasts
Botta et al. [60]	Orbital tissue from 5 GO patients Normal orbital tissue from 5 patients who underwent eye surgery for unrelated reasons	Enalapril Fibroblasts were treated with enalapril or as a control, with lisinopril Cell proliferation, lactate dehydrogenase release (as a measure of cell necrosis), apoptosis, and HA in the cell media were measured	Reduction of cell proliferation and HA release in GO fibroblasts: the proliferation of OF was reduced in both GO and control fibroblasts by enalapril; because enalapril did not affect necrosis or apoptosis, the effects on proliferation probably reflected an inhibition of cell growth and/or a delay in the cell cycle; enalapril reduced HA in the media from both GO and control fibroblasts Lisinopril had negligible effects
Rotondo Dottore et al. [47]	Orbital adipose tissue from 6 GO patients Normal orbital tissue from 6 patients who underwent eye surgery for unrelated conditions	Selenium To induce oxidative stress, cells were incubated with H2O2 at various concentrations; to assess the effects of selenium, cells were preincubated with medium without compounds or with medium containing selenium (SeMCys hydrochloride) or MCys Cell proliferation, HA, and pro-inflammatory cytokines production were measured	Reduction of proliferation, release of HA and cytokines in GO fibroblasts: H2O2 induced an increase in cell GSSG (a marker of oxidative stress) and fibroblast proliferation, which were reduced by selenium; H2O2 promoted production of the cytokines involved in the response to oxidative stress, i.e., TNF-α, IL1-β, and IFN-γ; the increase in TNF-α and IFN-γ was rescued by selenium; the effects of selenium were similar in GO and control fibroblasts concerning oxidative stress and cytokines, i.e., they were exclusive to GO fibroblasts concerning proliferation and HA
Rotondo Dottore et al. [58]	Orbital adipose tissue from 6 GO patients Normal orbital tissue from 6 patients who underwent eye surgery for unrelated conditions	Selenium Cells were incubated with H2O2 at 50 µM To assess the effects of selenium, cells were preincubated with medium without compounds or with medium containing SeMCys or, as a control, MCys Cell vitality, lactate dehydrogenase production (as a measure of cell necrosis), and apoptosis were measured	Reduction of cell damage in GO fibroblasts: SeMCys, rescued from H2O2-dependent cytotoxicity, by reducing necrosis and apoptosis, with no difference between GO and control fibroblasts; MCys had no effect; H2O2 determined a significant increase in GSSG, which was counteracted by SeMCys, but not by MCys, with no differences between GO and control fibroblasts
Rotondo Dottore et al. [61]	Orbital adipose tissue from 6 GO patients Normal orbital tissue from 6 patients who underwent eye surgery for unrelated conditions	Vitamin C, N-acetyl-cysteine, and melatonin Cells were treated with H2O2 to induce oxidative stress; cell vitality assays were performed to determine the noncytotoxic dose of each antioxidant; the following assays were performed: GSSG, cell proliferation, HA, TNF-α, IFN-γ, and IL-1β	Reduction of cell proliferation and HA release in GO fibroblasts: all of the 3 antioxidant agents reduced H2O2-dependent oxidative stress; vitamin C reduced proliferation in GO but not in control fibroblasts. N-acetyl-L-cysteine reduced proliferation and IFN-γ in GO, and HA and IL-1β in both GO and control fibroblasts; melatonin reduced IL1β and HA in GO and control fibroblasts, and IFN-γ only in GO fibroblasts
Rotondo Dottore et al. [67]	Orbital adipose tissue from 6 GO patients Normal orbital tissue from 6 patients who underwent eye surgery for unrelated conditions	Retinol, β-carotene, and vitamin E Oxidative stress was induced by incubation with H2O2; to assess the effects of the various antioxidant agents, cells were preincubated with complete medium without compounds or with medium containing either one of the compounds at various concentrations GSSG, cell proliferation, hyaluronic acid, TNF-α, IFN-γ, and IL-1β were measured	Reduction of cell proliferation in GO fibroblasts: all of the 3 antioxidants reduced the increase in GSSG induced by H2O2 in GO but not in control fibroblasts; β-carotene reduced the increased proliferation induced by H2O2 in GO but not in control fibroblasts, whereas retinol and vitamin E had no effect; IL-1β was reduced by all 3 substances; retinol reduced IFN-γ in GO and control fibroblasts