The effect of 2,2′-dichlorodiethyl sulfide on DNA synthesis of a murine stratified keratinocyte culture system

Abstract

A primary stratified keratinocyte culture resembling the epidermis in situ was used as a model for studying the effects of exposure to 2,2'-dichlorodiethyl sulfide, or sulfur mustard (SM), on DNA synthesis. A method that distinguishes between semi-conservative (s.c.) DNA synthesis and repair synthesis was used to determine if the former was inhibited following treatment with SM. In this method the density of the newly synthesized DNA was increased by incorporation of 5-bromo-2-deoxyuridine. Density gradient centrifugation was then used to isolate the heavy DNA for quantification. It was demonstrated that topically applied SM in the dose range of 1-10 nmole/cm2 inhibited s.c. DNA synthesis (replication) in a dose and time related manner. Inhibition of DNA replication by SM would result in inhibition of cell division which must be preceded by s.c. DNA synthesis. This failure to replace damaged germinative cells may lead to the destruction of the basal layer which is observed in vivo and in our epidermal culture following exposure to SM. This may also be related to development of vesication observed in exposed intact human skin.

Dna Repair in Primary Human Keratinocyte Cultures After Low Level Exposure to Bis(2-Chloroethyl)sulfide

“…Any radioactivity found in the light, parental DNA peak should be the result of DNA repair, while any radioactivity found in the more dense fraction, containing BUdR, should represent DNA replication. An increase in buoyant densities does not occur in the parental peak due to the low amount of BUdR that would be replaced (Roberts et al, 1968;Legator & Flamm, 1973;Hennings & Michael, 1976;Zaman-Saroya et al, 1992). In order to clarify the parental peak, in some experiments the peak DNA content fraction, as measured by A 260 , was rebanded in a second and sometimes a third CsCl gradient.…”