pH-dependent toxicity of sulphur mustard in vitro.
The dependence of sulphur mustard (HD) toxicity on intracellular (pH(i)) and extracellular pH was examined in CHO-K1 cells. HD produced an immediate and significant concentration-dependent decline in cytosolic pH, and also inhibited the mechanisms responsible for restoring pH(i) to physiological values. The concentration-response of HD-induced cytosolic acidification, closely paralleled the acidification of the extracellular buffer through HD hydrolysis. A viability study was carried out in order to assess the importance of HD-induced cytosolic acidification. Cultures were exposed to HD for 1 h in media that were adjusted through a pH range (pH 5.0-10), and the 24 h LC(50) values were assessed using the viability indicator dye alamarBlue. The toxicity of HD was found to be dependent on extracellular pH, with a greater than eight-fold increase in LD(50) obtained in cultures treated with HD at pH 9.5, compared to those treated at pH 5.0. Assays of apoptotic cell death, including morphology, soluble DNA, caspase-3 activity and TUNEL also showed that as pH was increased, much greater HD concentrations were required to cause cell death. The modest decline in HD half-life measured in buffers of increasing pH, did not account for the protective effects of basic pH. The early event(s) that HD initiates to eventually culminate in cell death are not known. However, based on the data obtained in this study, we propose that HD causes an extracellular acidification through chemical hydrolysis and that this, in both a concentration and temporally related fashion, results in cytosolic acidification. Furthermore, HD also acts to poison the antiporter systems responsible for maintaining physiological pH(i), so that the cells are unable to recover from this insult. It is this irreversible decline in pH(i) that initiates the cascade of events that results in HD-induced cell death.
Sawyer
Thomas W
TW
Chemical Biological Defence Section, Defence Research and Development Canada - Suffield, Box 4000, Medicine Hat, Alberta, Canada T1A 8K6. Thomas.Sawyer@drdc-rddc.gc.ca
Vair
Cory
C
Nelson
Peggy
P
Shei
Yimin
Y
Bjarnason
Stephen
S
Tenn
Catherine
C
McWilliams
Michael
M
Villanueva
Mercy
M
Burczyk
Andrew
A
D000222:Adaptation, Physiological / Q000187:drug effects; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D053148:Caspase 3 / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D002619:Chemical Warfare Agents / Q000633:toxicity*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D045604:Extracellular Fluid / Q000737:chemistry / Q000187:drug effects; D005260:Female; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D007424:Intracellular Fluid / Q000737:chemistry / Q000187:drug effects; D009151:Mustard Gas / Q000633:toxicity*; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects*
D002619:Chemical Warfare Agents; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D053148:Caspase 3; D009151:Mustard Gas; C039614:ethylisopropylamiloride