[Human trabecular cells and apoptosis: in vitro evaluation of the effect of betaxolol with or without preservative].
OBJECTIVE
Trabecular meshwork, which is involved in aqueous outflow resistance, is deeply modified in glaucoma patients, with a decrease in the trabecular cell number. Trabecular toxicity of antiglaucoma medications cannot be excluded. On a human cultured trabecular cell line, we investigated the potential proapoptotic effect of a beta-blocker with or without preservative, benzalkonium chloride (0.01% BAC), by flow cytometry and confocal microscopy.


METHODS
and


METHODS
A human immortalized trabecular cell line (HTM-5) obtained from a normal donor was cultured under normal conditions. Preserved 0.25% betaxolol suspension (betaxolol BAC +), unpreserved 0.25% betaxolol suspension, and 0.01% BAC were respectively added to the culture medium in a 1/10 or 1/100 dilution for 15 minutes. After a 24-hour recovery period in normal culture conditions, cell size and the expression of an apoptotic marker, Apo 2.7, were evaluated by flow cytometry and confocal microscopy. Untreated trabecular cells were used as control cells.


RESULTS
Preserved and unpreserved betaxolol in a 1/10 dilution induced a significant decrease in trabecular cell size compared to controls. However, this cell size decrease was less pronounced than that induced by BAC at the same dilution. Similar results were obtained with betaxolol and BAC in a 1/100 dilution. Trabecular cell Apo 2.7 expression was significantly increased after treatment with betaxolol BAC + and BAC- in a 1/10 dilution compared to controls (36.8%, 28.1%, and 15.4%, respectively p<0.005). However, this proapoptotic activity was much less pronounced than that induced by BAC- at the same dilution (96.9%, p<10(-4)). Unpreserved betaxolol in a 1/100 dilution had no apoptotic activity on trabecular cells. Trabecular cell Apo 2.7 expression slightly increased with betaxolol BAC + at a 1/100 dilution (24.9%, p=0.04), while it was greatly increased with BAC at the same dilution (39.9%; p<10(-4)).


CONCLUSIONS
In our model, unpreserved betaxolol at a low concentration displayed no proapoptotic activity on trabecular cells. On the other hand, preserved betaxolol displayed a moderate proapoptotic activity by triggering cell death of around 25% of cells. Trabecular cell toxicity appeared to be mainly due to the preservative benzalkonium chloride (BAC). Taken together, our results demonstrated that the strong apoptotic activity of BAC was greatly reduced within the preserved eye drops, probably through the interaction of BAC with the active compound.
Hamard
P
P
Services d'Ophtalmologie, C.H.N.O. des Quinze-Vingts, Paris et Hôpital Ambroise Paré, APHP, Boulogne, France. pascale-hamard@quinze-vingts.fr
Debbasch
C
C
Blondin
C
C
Brignole
F
F
Loison-Dayma
K
K
Warnet
J M
JM
Baudouin
C
C
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D017209:Apoptosis; D001548:Benzalkonium Compounds / Q000494:pharmacology*; D015784:Betaxolol / Q000494:pharmacology*; D016923:Cell Death; D002460:Cell Line; D048429:Cell Size; D003470:Culture Media; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology* / Q000187:drug effects*
D000319:Adrenergic beta-Antagonists; D001548:Benzalkonium Compounds; D003470:Culture Media; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D015784:Betaxolol