Sulfur mustard (SM1
, bis(2-chloroethyl)-sulfide) is well-known as a chemical warfare agent that causes acute cutaneous toxicity, as well as ocular and pulmonary toxicity (1
). Chemically, SM reacts via an electrophilic episulfonium intermediate (2
) and directly damages DNA and other macromolecules. The major identified DNA adducts are N7-(2-hydroxyethylthioethyl)-guanine, N3-(2-hydroxyethylthioethyl)-adenine and a crosslinked product, di-(2-guanin-7-yl)ethyl sulfide, which accounts for only 10-20% of the total adducts (5
). A monofunctional analog of SM, 2-chloroethylethylsulfide (CEES) forms analogous N7-guanine and N3-adenine adducts, but does not form cross-links with DNA (7
). In vitro
studies utilizing cells that genetically lack the ability to repair these adducts have provided strong evidence that DNA damage is the major determinant of cytotoxicity due to these sulfur mustards. Both nucleotide excision repair and base excision repair have been implicated in the repair of the monoadducts (8
), while repair of the crosslink also involves homologous recombination.
There is a general correlation between the ability of toxic agents to damage DNA, and their ability to induce mutations and cause cancer. Indeed, epidemiological studies of mustard gas workers in the UK found significantly elevated risk of cancer of the upper aerodigestive tract (10
). Cancers of the larynx, pharynx and oral cavity were two-five times more common in the mustard gas exposed population than expected, and lung cancer was also significantly elevated. Similarly, in studies of former mustard gas workers in Japan, deaths from cancers of the respiratory tract were over 30-fold higher than expected (11
It has long been appreciated that the unifying aspect of most chemical carcinogens is their ability to either act directly as electrophiles, or be metabolized to electrophilic intermediates. Thus, chemical strategies for scavenging electrophilic carcinogens may be expected to prevent the induction of DNA damage by the sulfur mustards and thereby block toxicity. One such successful strategy utilized thiopurines as nucleophilic trapping agents for the electrophilic ultimate carcinogen BPDE (12
). Initial studies in CHO cells demonstrated that pretreatment with 6-mercaptopurine (6MP) could completely block the ability of BPDE to form covalent adducts in cells, with a corresponding reduction of BPDE-induced toxicity and mutation frequency (14
). This was correlated with the intracellular formation of the expected adduct between 6MP and BPDE. Part of the reason for this exceptional activity is that thiopurines are substrates for the cellular purine transport system (15
), allowing rapid accumulation of the scavenging agent intracellularly. However, 6MP is a cytotoxic anti-cancer agent; toxicity is due to its incorporation into DNA as a purine base. Other thiopurines, in particular 2,6-dithiopurine (DTP), are not converted into nucleotides in mammalian cells and therefore do not have this cytotoxic activity.
Chemically, DTP, thiopurinol, 9-methyl-6-mercaptopurine (MMP), 6-thioxanthine (6TX) and 2,6-dithiouric acid (DUA) were shown to react facilely with BPDE and several other electrophilic carcinogens (13
). These studies were extended in a mouse model of skin carcinogenesis in which the carcinogenic process was initiated with topical application of an initiating dose of BPDE to the shaved dorsal skin, followed by twice weekly application of the tumor-promoting agent TPA. This results in the formation of multiple papillomas per mouse over the course of ~20 weeks, and the ultimate conversion of a fraction of the lesions to squamous cell carcinomas. Topical application of DTP to the dorsal skin 15 min prior to BPDE treatment resulted in a dose-dependent reduction in both papilloma incidence and multiplicity, and in carcinoma incidence (17
). The extent of reduction in tumor formation closely matched the reduction in the formation of BPDE-DNA adducts in the treated epidermis, with 90-95% reduction of all parameters at the higher dose of DTP.
We have recently found that DTP reacts facilely with two monofunctional analogs of sulfur mustard, 2-chloroethyl ethyl sulfide (CEES), and 2-chloroethyl methyl sulfide (CEMS) (accompanying ms.). In those in vitro studies, DNA was not able to compete effectively with DTP for CEMS reaction. Since there is good reason to expect that preventing DNA damage should block both cytotoxicity and mutation induction, we hypothesized that DTP might provide protection from CEES- and CEMS-induced cytotoxicity in cells by scavenging the reactive toxicant before any cellular damage is produced. In the present study we show that DTP can block the cytotoxic and mutagenic effects of CEES and CEMS in human skin cells.