Macromolecular metabolism of a differentiated rat keratinocyte culture system following exposure to sulfur mustard

Abstract

A method for producing a stratified, squamous epithelium in vitro by cultivating rat keratinocytes on nylon membranes has been developed in this laboratory. This epidermal-like culture is being used to obtain a better understanding of the mechanism of skin vesication after topical exposure to the sulfur mustard bis(beta-chloroethyl) sulfide (BCES) dissolved in a selected solvent. Radiolabeled macromolecular precursors (thymidine, uridine, and leucine) have been used to study the effect of BCES on the synthesis of DNA, RNA, and protein, respectively, after topical exposure to the mustard at concentrations of 0.01-500 nmol/cm2 dissolved in 70% dimethyl sulfoxide (DMSO). From these and other studies it has been determined that exposure to even the low concentration of 0.01 nmol BCES/cm2 for 30 min results in significant inhibition of [3H]thymidine incorporation, although complete recovery occurs by 24 h. Significant inhibition of [3H]uridine and [14C]leucine incorporation is observed only after exposure to much higher concentrations of BCES (10-500 nmol/cm2). This suggests a very early lesion in macromolecular metabolism with DNA being the primary target.

Nitrogen mustard inhibits transcription and translation in a cell free system

“…This suggests that transcription is a more readily hindered process than translation and hence DNA is probably the more sensitive target for the mode of action of mustards. This conclusion is consistent with other studies involving mustard effects in situ (13)(14)(15)(16)). This may reflect either the differences in rates of mustard alkylation of the DNA and RNA templates (which are currently not known), or the differences in the response of RNA polymerase to alkylated DNA template compared to the response ribosomes to an alkylated RNA template (or a combination of both effects).…”

“…Earlier work demonstrated that a higher concentration of sulfur mustard was required to inhibit RNA and protein synthesis than was required to inhibit DNA synthesis (13,14). Similar conclusions were also drawn from other studies using human and rat keratinocytes in cell culture (15,16). These studies therefore suggest that DNA is the most sensitive nucleic acid target of mustards.…”

Mustard gas crosslinking of proteins through preferential alkylation of cysteines

“…Many recent studies have focused on the reactivity and crosslinking of DNA and RNA with mustard (Pechura and Rall, 1993;Papirmeister et al, 1985;Gross et al, 1985). However, mustard reactivity with nucleotides alone appears insufficient to explain all of the effects of mustard exposure (Watson and Griffin, 1992;Vaughan et al, 1988). It has long been known that mustard is capable of modifying proteins (Watson and Griffin, 1992;Kirner, 1946;Hambrook et al, …”

Pseudoepidermis, Constructed in vitro, for Use in Toxicological and Pharmacological Studies

“…Primary cultures of rat kératinocytes were pro duced by a procedure described in previous reports [3,7], Kératinocytes were isolated from the back skin of newborn rats (1-2 days old). Animals, obtained from a colony maintained in this laboratory by ran dom breeding for over 15 years, were sacrificed by cervical dislocation.…”

“…This cul ture undergoes terminal differentiation and displays morphological [1] and biochemical [2] markers of the parent tissue. The system has been used for studying epidermal differ entiation and homeostasis and for investi gating the effects of environmental agents on those factors that influence normal epider mal function [3].…”