In vitro assays
Bacterial reverse mutation test (AMES test)
In vitro mammalian cell gene mutation test
Escherichia coli, reverse assay
In vitro mammalian chromosome aberration test
Sex-linked recessive lethal test in Drosophila melanogaster
In vitro sister chromatid exchange assay in mammalian cells
Comet assay
DNA damage and repair, unscheduled DNA synthesis in mammalian cells in vitro
In vitro mammalian cell micronucleus test
HPRT (Hypoxanthine phosphoribosyltransferase) assay
Mouse lymphoma assay
In vivo assays
Mammalian erythrocyte micronucleus test
Mammalian bone marrow chromosome aberration test
Mammalian spermatogonial chromosome aberration test
Comet assay
Unscheduled DNA synthesis
Following genetic damage, cells undergo DNA repair mechanisms in the form of gene mutation, recombination or chromosomal damage. Aneuploidy and larger scale numerical chromosomal damage are of vital genetic changes and might have been associated with malignancy (Weaver et al. 2007). Compounds that detect such kind of damage and are positive in genotoxicity tests are considered to be potential carcinogens (Lichtfouse et al. 2012). There is a confirmed relationship between exposure to particular chemicals in humans and carcinogenesis. Genotoxicity tests have been almost used for cancer prediction. Therefore the outcome of genotoxicity tests can be valuable for the interpretation of carcinogenicity studies. Mutations are usually associated with human diseases.
Genotoxicity tests are usually performed in bacterial, yeast, and mammalian cells and the findings would help us to control and improve the cellular defense against genotoxic substances (Kolle Susanne 2012).
12.3 Genotoxicity of Sulfur Mustard
Sulfur Mustard is regulated under the Chemical Weapons Convention (CWC) among the classes of chemicals which monitored under the highest risk class (Ganesan et al. 2010). Although sulfur mustard may be lethal in higher doses, it usually causes extensive acute and chronic injuries in different organs. LC t50 (lethal concentration-time product) of SM for humans is 900 mg-min/m3 for 2–10-min exposures (NRC 1997). Sulfur Mustard (SM) and its analogs are of the first chemical agents which their genotoxic and mutagenic effects has been confirmed (Fox and Scott 1980). SM is responsible for over 80 % of all chemical injuries which have been reported and the most recent use of SM was in Iran-Iraq war (Chilcott et al. 2000).
In vitro studies in prokaryotic organisms (Salmonella typhimurium and Escherichia coli) and eukaryotic organisms (HeLa cells, mouse lymphoma, and rat lymphosarcoma) are among the first studies which propose the genotoxicity of sulfur mustard. DNA cross-links formation, DNA alkylation, inhibition of DNA synthesis and repair, point mutation, and chromosome aberration formation were suggested mechanisms. Increasing the frequencies of chromosomal aberration in a dose dependant manner and mutation induction in HPRT (hypoxanthine guanine phosphoribosyl transferase) test are of the first genotoxic studies (Jostes et al. 1989).
Low doses of SM induce DNA cross links and thus replications and repair errors in DNA, which may cause mutation (Papirmeister et al. 1991). In a study on rat epidermal keratinocytes cultures exposed to SM, a dose related interstrand crosslink of DNA has been confirmed. These cross links effects on DNA synthesis and induces cell cycle block (Lin et al. 1996). Another study showed a mismatch repair in DNA bases of monkey kidney cells following exposure to SM (Fan and Bernstein 1991). SM causes DNA alkylation in a bacteriophage and the most common sites of DNA alkylation were on 5′-AA, 5′-GG, and 5′-GNC sequences on the DNA template strand. SM at the doses of 0.5–0.1 mM produced single strand breaks (Venkateswaran et al. 1994).
In-vivo studies in Drosophila showed that SM injection caused point mutation in male flies (Auerbach et al. 1947). Positive micronucleus test in mouse bone marrow exposed to sulfur mustard was also evidence of SM genotoxicity (Ashby et al. 1991). Ludlum exposed human white blood cells to labelled SM in vitro, and he measured a SM DNA adduct 7- (2-hydroxyethylthioethyl) guanine in cell culture media (Ludlum et al. 1994). Fishermen who were exposed to sulfur mustard shells, showed sister chromatid exchanges in their lymphocytes (Wulf et al. 1985). Emison observed DNA damage in human epithelial cell culture after exposure of the cells to SM. A cell cycle block was found at the G1-S and G2-M phases at the concentrations of below and equivalent of vesicating concentration of SM (100 μM) (Emison and Smith 1996).
12.3.1 Mechanisms of SM Genotoxicity
The most accepted theory of SM toxicity is alkylation reactions with DNA, RNA and proteins in cells. After absorption, SM comes in the form of an ionic intermediate, ethylene episulfonium. Ethylene episulfonium cation undergoes intramolecular cyclisation and transforms to a very active carbonium ion. Carbonium ion rapidly reacts with nucleophiles such as DNA and a large number of electron- rich molecules such as sulfhydryl and amine groups of proteins and nucleic acids (Wheeler 1962).
SM induces DNA adducts and cross links between and inside DNA strands and causes DNA breaks and inhibition of protein synthesis (Walker 1971). Thus, the results are creating abnormal chromatids and inhibition of DNA, RNA and protein synthesis. The main DNA alkylation occurs on the N7-position of guanine (Kehe and Szinicz 2005).
Cross links and adducts constitute 15 % and 85 % of DNA damages respectively, but the cytotoxicity of SM is related to cross links which prevent DNA replication (Matijasevic et al. 2001). DNA damage by SM exposure activates poly (ADP-ribose) polymerase-1 (PARP-1) and stimulates several DNA repair pathways, including base excision repair, nucleotide excision repair, and homologous recombination. If this genotoxic stress cannot be repaired, the cell will start the apoptotic program (Jowsey et al. 2012).
DNA strand breaks activate DNA repair enzymes; especially poly ADP ribose polymerase (PARP) and this reduce nicotinamide adenine dinucleotide (NAD) resources in cells. ATP is also used for the synthesis of NAD and this caused a reduction in the cellular pools of ATP and disruption in the supply of cell energy (Lindahl 1979). Alkylation and inactivation of sulfhydryl-containing proteins and peptides such as glutathione is the other mechanism of cell death. These proteins are crucial in stabilizing the oxidation redox position of cells (Maynard 1995).
12.4 Mutagenicity of Sulfur Mustard
Several studies have documented the mutagenic effects of SM in mammalian cells, in vivo and in vitro test systems (Papirmeister et al. 1991). An aims assay on salmonella with tester strains TA97, TA98, TA 100 and TA102 at the concentrations of 0.01–250 μg per plate of SM, was not able to show the mutagenic response by any of the strains (Stewart et al. 1989). However, The mutagenic properties of mustard compounds have been confirmed in other organisms including Ecoli and Neurospora by Horowitz and Tatum (Horowitz et al. 1946; Tatum 1947). SM is mutagens in diverse assays, including ames tests for germ cell mutations in drosophila and dominant lethal in mice and in salmonella TA97a and TA102 strains (Vijayan et al. 2014).
Two major reasons of mutation induced by SM are point mutations (mis-matched) and mutations in repair enzymes (mis-repair). DNA repair enzymes enter a base in the damaged area and in front of alkylating purines, but if the base is incorrectly inserted, may cause errors during DNA replication and mutations. Mutation in tumor suppressor genes or oncogenes, causes uncontrolled cell proliferation. For example, mutations in p53 in Japanese workers at factories produced mustard gas have been reported (Yanagida et al. 1988). In a survey on lung tumors of workers who had worked in SM factory, p53 mutations were found which were the similar to mutations in lung tumors of tobacco smokers except with the prominence of double mutations in workers of SM factory (Takeshima et al. 1994).
Lung cancer biopsies from Iranian patients, who had a single exposure to SM during Iran-Iraq conflict, have been analyzed. DNA was extracted from the tumor tissue, PCR amplified and sequenced to detect p53 mutation. Eight p53 mutations with two double p53 mutations have been observed and the dominant site of mutations was G to A (Hosseini-khalili et al. 2009).
12.5 Carcinogenicity of Sulfur Mustard
SM is categorized as a carcinogen (IARC 1994) and several epidemiologic studies provide sufficient evidence that SM is carcinogenic in humans, particularly in the upper respiratory tract. Although there is not a dose–response relationship in carcinogenicity of SM in human studies, laboratory studies have shown a relationship between SM and respiratory cancers, skin cancers and leukemia (Pechuta and Rall 1993).
In a study, male and female strain A mice exposed to SM through breathing for every 15 min showed a significantly higher incidence of lung cancers than their controls with no SM exposure (Heston 1953). Another study on guinea pigs, mice, rabbits, and dogs that were exposed to sulfur mustard in the air for 3–12 months did not reveal any cancers except for the squamous cell carcinoma (SCC) in rat’s skin (McNamara et al. 1975).
Occupational studies of Japanese and British workers, who manufactured SM, have shown higher incidence of respiratory cancers compared to normal populations. British factory workers who had manufactured SM, had a significant rate of death from larynx, pharynx, lung, mouth, esophagus and stomach cancer compare with death in the normal population (Easton et al. 1988). In Japanese workers, the number of deaths from cancers of the respiratory tract were higher compared to fatality expected from such cancers (37 vs. 0.9 respectively) (Wada et al. 1968). Another follow up study in Japanese factory workers was performed 17–20 years after 7–9 years of exposure to SM. Of all the reported deaths, 28 % were because of cancers compared with 7.7 and 8.5 % in two groups of unexposed residents of the same area. The most common types of cancers were squamous cell carcinoma and small cell carcinoma (Yamada 1963). Nishimoto Investigated 2068 Japanese factory workers. Among the workers, those who had the highest SM exposure had three times more deaths of cancers compared to the area male population (Nishimoto et al. 1983). The same study was performed on German factory workers who manufactured SM. In a 20 year follow up, malignant bronchial carcinoma, leukemia and bladder carcinoma were significantly increased (Weiss and Weiss 1975). An epidemiologic study of World War 1 veterans who were exposed to SM was done 15 years after their exposure. This study revealed that the number of deaths due to lung cancer was doubled compared to controls (Case and Lea 1955). In another study on American veterans of World War 1, the incidence of cancers of upper respiratory tract was slightly higher than control (Beebe 1960). Nasopharynx carcinoma, bronchogenic carcinoma, gastric adenocarcinoma, ALL (Acute lymphoblastic leukemia) and AML (Acute myeloid leukemia) have been reported in chemically injured Iranian veterans with SM (Ghanei and Vosoghi 2002). However Emad and Rezanian in a study of 197 Iranian veterans 10 years after acute SM toxicity in the Iran-Iraq conflict couldn’t show any upper respiratory tract malignancies (Emad and Rezaian 1997).
12.6 Teratogenicity of Sulfur Mustard
Few studies are available regarding the reproductive effects of SM in animals and humans and the results are controversial. Intravenous injection of SM in male mice causes a transient damage to the testes and inhibition of spermatogenesis with a full recovery 4 weeks after exposure (Graef et al. 1948). Another study in mice who were receiving SM intraperitoneal during the gestation period, different types of birth defects, including craniofacial and septal defects as well as the limb malformations was observed (Sanjarmoosavi et al. 2012). SM exposure in rats who gavaged by different doses of SM did not reveal any significant damage on fertility and reproductive activities in two generations study (Sasser et al. 1996). In a study in male rats, exposure to 0.1 mg/m3 of SM 5 days/week for up to 52 weeks significantly increased the rate of lethal mutations in somatic and germ cells (9.4 % in SM compared to 3.9 % in controls) (Rozmiarek et al. 1973).
In Iranian veterans with exposure to SM, in the first 5 weeks after exposure, the level of testosterone has been decreased with an increase in FSH and LH, however all hormones had returned to normal after 12 months. Of those veterans, (29 %) had decreased sperm count below 20 million. In a testicular biopsy performed on 50 % of men with sperm count below two million cells per ml., complete or relative arrest of spermatogenesis was confirmed (Azizi et al. 1995). Another study on Iranian SM veterans 3–9 years after exposure also showed significant reduction in the number of sperms and motility of sperms compared to healthy controls (Balali-Mood and Hefazi 2005). On the other hand, in a 12 month survey following SM exposure in a group of SM exposed veterans, the incidence of infertility was almost close to this number for a worldwide average (Ghanei et al. 2004). Another study in Iranian SM veterans reported a significant increase in the rate of fetal deaths and congenital malformations in children who were borned after single exposure to SM compared to control (Pour-Jafari et al. 2011).
12.7 Application of Laboratory Tests in Evaluation of Genotoxicity of Sulfur Mustard
12.7.1 Measurement of DNA Damage Induced by Sulfur Mustard
The most important mechanism of SM pathogenesis is the reaction of SM with DNA which creates DNA mono adducts or cross links (Ashby et al. 1991). DNA mono adducts are thought to be more related to delayed genotoxicity of SM and result to mutations in cells, which survive from SM toxicity (Jowsey et al. 2012). Cross links induce DNA double strand breaks during DNA replication, and activate DNA repair enzymes such as poly (ADP-Ribose) polymerase-1 (PARP-1) (Papirmeister et al. 1991). Thymocytes which exposed to different concentrations of SM over a period of 24 h, showed an increased level of DNA fragmentations followed by laddering pattern suggesting apoptosis (Michaelson 2000). Another study showed a dose-dependent increase in DNA damage in TK6 lymphoblastoid cells incubated with a SM analogue, CEES (2-chloroethyl ethyl sulfide) (Jowsey et al. 2009).
Comet assay is a rapid and sensitive test to detect DNA damage in vitro. A modified comet assay technique using DNA repair enzymes formamido-pyrimidine-gly-cosylase (FPG), endoglycosylase III (ENDO III) and 3-methyladenine-glycosylase (AAG) was able to show SM induced DNA damage in pigs’ skin cells. Repair enzymes increase the sensitivity of the comet assay and are able to detect DNA damage at the SM concentration of 30 nmol/L (Kehe et al. 2009). A study on Iranian veterans using comet assay 20 years after exposure to SM showed DNA damage in DNA lymphocytes, which was significantly higher than non SM exposed controls. Mutations in DNA repair genes of the hematopoietic cells at the time of the initial exposure are a possible explanation for the delayed DNA damage. The other suggested mechanism of such finding is DNA damage due to a general inflammatory/ oxidative stress mechanism (Behravan et al. 2013).
12.7.2 Evaluation of Proteins Involved in DNA Damage Signalling in Sulfur Mustard Toxicity
DNA damage signalling cascades orchestrates through ATM (ataxia telangectasia mutated) and ATR (ataxia telangectasia related) protein kinases. These kinases respond to different types of DNA damage. For example, ATM, is activated following DNA double strand breaks (DSB) while ATR is activated by different types of DNA damage, including DNA cross links and adducts (Hurley and Bunz 2007). Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2 (Chk2) regulate cell functions such as DNA replication and cell cycle progression or apoptosis. There are common substrates for both Chk1 and Chk2 and combination of these 2 molecules has been documented (McGowan 2002). Some Chk1 and Chk2 effectors can be categorized as tumor promoter or tumor suppressor genes (Bartek and Lukas 2003). Activated ATR and ATM, phosphorylated many target proteins, including checkpoint kinases (Chk1, Chk2) and p53. Chk2 mainly activated by ATM in response to DNA double strand breaks (DSBs). Chk1 is believed to be associated with the ATR, however cross connection with ATM has also been seen (Gatei et al. 2003).
P53 is a tumor suppressor protein and is activated following cellular stress, such as DNA damage and hypoxia. This protein causes cell cycle arrest or apoptosis, to inhibit malignant transformation of cancer cells. The lack of a normal p53 protein, allows the mutations to accumulate and create a tumor(Ghosh et al. 2004). These proteins have a vital role in preventing genetic lesions by slowing down the cell cycle, regulating the transcription and increasing the power of DNA repair in cells (Ljungman 2005). An in vitro study on lymphoblastiod cell line exposed to sulfur mustard demonstrated the dose- and time-dependent activation of DNA damage signalling pathways, in particular the phosphorylation of CHK1, CHK2 and p53 (Jowsey et al. 2012).
12.7.3 Evaluation of Proteins Involved in DNA Repair Signalling in Sulfur Mustard Toxicity
To have a better understanding of sulfur mustard toxicity and to provide a treatment, we should increase our knowledge about the mechanism that cells utilize to protect against sulfur mustard damage. Simple DNA adducts and lesions caused by oxidative stress, such as methylation are repaired by base excision repair pathway (BER). A DNA repair enzyme known as PARP-1 (Poly (ADP-ribose) poly merase-1) plays a critical role in the BER pathway. Cells lacking PARP-1 protein are very sensitive to chemicals which induce DNA alkylation (Dantzer et al. 2000). Bulky DNA adducts, such as DNA cross linking are repaired by NER (Nucleotide excision repair) (Jowsey et al. 2009).
Severe DNA damage induced by SM decreases NAD and cell repair restoration and cellular ATP (Burkle 2001). While the small DNA lesions activates DNA repair pathways and caused DNA repair, severe DNA injuries caused cell apoptosis and cell death.
Rad proteins are important DNA repair checkpoints which arrest cell cycle progression at early stage of DNA damage. These proteins ensure the transmission of undamaged genetic material to daughter cells (Abraham 2001). The DNA repair signalling pathway was studied in mouse liver percutaneously exposed to SM. DNA repair proteins Rad23, Rad50, Rad51, Rad52, and Rad54l were decreased during a week after exposure and results indicated that SM promotes DNA double strand breaks (DSB) which caused cell death(Anand et al. 2009). Incubation of TK6 lymphocytes with SM and studying DNA repair pathways showed that homologous recombination (HR) is the major repair cascade protecting against acute SM toxicity while NER has also positive effects in this pathway (Jowsey et al. 2012). A host cell repair assay in Chinese hamster ovary cells showed that nucleotide excision repair (NER) involves in repairing DNA damage caused by SM and decreases SM toxicity (Matijasevic et al. 2001).
12.7.4 Measurement of Oxidative Stress in Sulfur Mustard Toxicity
The cytotoxicity of SM has been proposed to result from a series of alkylation reactions and production of reactive oxygen substances (ROS). After absorption of SM into the body, it forms the highly reactive carbonium ion which reacts with DNA, proteins and other molecules such as glutathione. Glutathione depletion increases the level of ROS production (Kehe and Szinicz 2005). Also, ROS are changed into highly toxic oxidants that cause membrane phospholipids to form lipid peroxides, leading to loss of membrane function. Over stimulation of poly (ADP ribose) polymerase (PARP) following SM induced DNA damage also leads to consumption of cell energy and generation of reactive oxygen species (Korkmaz et al. 2008). In a study, antioxidant enzyme activities were measured 24 h after dermal exposure of rat with SM. As a result of glutathione and NAD depletion, glutathione peroxidise activity decreased significantly in white blood cells, spleen and liver (Husain et al. 1996). Another study in mice after 12 weeks of chronic exposure to SM showed increased lipid peroxidation and reduced levels of antioxidant enzymes, glutathione reductase and glutathione peroxidise (Sharma et al. 2009). In human acute SM toxicity induces oxidative stress and decreases the glutathione reserves (Balali-Mood and Hefazi 2006).
There is a direct relationship between SM toxicity and oxidative stress. Antioxidant therapy in the protection and treatment of SM poisoning has been proposed previously. Various studies in laboratory animals have been shown the protective effects of antioxidants in SM toxicity (Gautam et al. 2007; Pohanka et al. 2011, 2013). Also, some studies have shown oxidative stress in Iranian veterans who were exposed to SM and a significant decrease in the activities of some antioxidant enzymes has been found (Shohrati et al. 2010).
12.7.5 Evaluation of Chromosomal Aberration in Sulfur Mustard Toxicity
The incidence of chromosomal abnormalities caused by SM depends on the amount of primary alkylation, deletion prior to DNA replication and cell repair capacity after DNA replication. Cross linking of DNA induced by SM causes chromosomal abnormalities and it is suggested that inter-strand cross links are more responsible for these abnormalities compared to intra- strand cross links. DNA cross- linking due to SM cause chromosomal aberration and although it is not observable until mitosis, the exact damage has been induced during DNA replication (Papirmeister et al. 1991). It has been seen that chromosomal damage in SM is dose dependent and end G1 and early S phases of the cell cycle are the most sensitive sites to SM damage (Savage and Breckon 1981). P53 protein has an important regulatory role in cell cycle and genetic stability and mutations in the p53 gene has already been discussed in SM toxicity. P53 mutation is considered to be an important cause of aneuploidy (Takeshima et al. 1994; Schmitt et al. 2002; Karami et al. 2007).
A significant increase in the incidence of sister chromatid exchanges has been reported in the peripheral lymphocytes of fishermen who were exposed to SM (Wulf et al. 1985). Rat lymphocyte cell line incubated with SM was examined for the evaluation of chromosomal damage. DNA and RNA alkylation and chromosomal aberration were found and the amount of damage was the same as chromosomal damage due to X-irradiation (Scott et al. 1974a, b).
Another study was performed on Iranian chemical veterans, 7 years after exposure to SM and it showed aneuploidy in the type of hyperdiploidy (22 of 27). All patients were classified as severe disability due to SM injury (Hassan and Ebtekar 2002). The results of the same study in a similar group of Iranian veterans revealed hyperdiploidy and Philadelphia chromosomes in bone marrow aspiration (Ghanei and Vosoghi 2002).
There have been many research studies on in vitro, in vivo and clinical impacts of sulfur mustard toxicity. In Tables 12.2, 12.3, 12.4, and 12.5 we reviewed them.
Table 12.2
In vitro studies on the genotoxicity of SM and its analogues
Compound (SM, NM, 2CEES) | Assay | Concentration/duration | Cell line | Results | Ref. |
|---|---|---|---|---|---|
SMa | Ames test (salmonella/microsome assay) | SM: 10 and 50 μg/plate | Salmonella strains (TA97a, TA98, TA100, TA102, TA104) in the presence and absence of S9 mix | Aminofostine analogs (chemical radioprotectors) decreased SM-induced mutagenicity | Vijayan et al. (2014) |
NMa | Comet assay, immunofluorescence, confocal microscopy, Western blot | NM: 0.75 μM/4–72 h | JB6 (mouse epidermal cells) | NM induced inter-strand cross-link, DNA double strand break, decreased cell growth and S-phase arrest Homologous recombination repair (HRR) showed as a key pathway involved in repair of NM-induced DNA Double strand break | Inturi et al. (2014) |
SM, CEESa | Western blot | SM: 0.1–1 μM CEES: 100–500 μM | TK6 lymphoblastoid cells, Fibroblast cells (GM04312 & GM15876) Chinese hamster ovary (CHO) cells (EM9, EM9-XH, V-C8 and V-C8 + B2) | Homologous recombination (HR) was the major repair pathway protecting against the acute SM toxicity with nucleotide excision repair (NER) and non-homologous end joining (NHEJ) also contributing to cell survival Dose and time-dependent activation of DNA damage signalling pathways was shown after SM exposure, in particular phosphorylation of Chk1, Chk2a and p53 | Jowsey et al. (2012) |
SM | Western blot, Immunofluorescence assay | SM: 1, 5, 20 or 100 μM for 24 h | Hela, Chinese hamster ovary cells (V-C8, V-C8 + B2) and lymphoblastoid cells (TK6) | DNA double strand breaks after SM exposure Cells lacking the homologous recombination DNA repair (HR) pathway were more sensitive to the SM toxicity Chemical activation of the HR protein offer cellular protection against SM | Jowsey et al. (2010) |
SM | UV/V is spectroscopy, Gel electrophoreses | SM: 25–1000 μM | Rat liver active (S1 and S2) and inactive (P2) chromatin | Unfolding of the chromatin was shown in concentration <500 μM of SM, at higher concentrations condensation of chromatin due to forming cross-links between the chromatin components was detected The incidence of condensation was higher in S2 phase | Jafari et al. (2010) |
SM | Neutral red uptake assay, XTT, Comet assay | SM: 0.12–250 μM | HeLa, A549, HepG2, AA8 | The LC50 (0.2 μM of SM) was associated with the lowest concentration that DNA cross-links were found The higher concentration (10 μM) of SM resulted in inhibiting the basal metabolism | Jost et al. (2010) |
CEES | Western blotting, comet assay, DNA adduct immunoassay | CEES: 0.2–1 mM | Lymphoblastoid cell lines (TK6, DK0064, LB707, LB708) | CEES induced dose-dependent increase in DNA damage via induction of P53 and Chk2 phosphorylation Also, the role of base excision repair (BER) and nucleotide excision repair (NER) pathways were shown in CEES-DNA damage repair | Jowsey et al. (2009) |
SM & CEES | Bacterial and cell survival, Host cell reactivation assay | SM: 50–200 mM CEES: 200–1000 mM | Bacteria: MV1161, wild type; MV1273, uvrA6; MV1174, alkA1; MV1302, alkA1 uvrA6 Mammalian cells: Mouse embryonic fibroblasts (MEF); wild type and 3-alkyladenine DNA glycosylase null mutant | The presence of a functional NERa pathway increased survival and reduced mutagenesis whereas the presence of a functional BERa pathway reduced survival, increased mutagenesis and decreased repair | Matijasevic and Volkert (2007) |
SM | TUNELa, Western blot | SM: 30–1000 mM for 30 min | Pulmonary A549 cells | SM induced DNA fragmentation and dose- dependent increase in PARPa after 24 h Also, increased AChEa activity was detected in SM-exposed cells | Steinritz et al. (2007) |
SM | RT-PCRa, Western blot, Spectrofluorometry, Hoechst staining | SM: 100, 200 and 300 μM | Primary human keratinocyte | The activation of calmodulin, calcineurin and Bad during SM-induced apoptosis in keratinocytes | Simbulan-Rosenthal et al. (2006) |
SM | Microarray | 200 μM for 2 h | Human epidermal keratinocytes | The transcriptional profile of SM compared with lewisite as a vesicant agent also with a genotoxic agent (Cisplatin) Apoptotic transcripts were found in Lewisite but not in SM | Platteborze (2005) |
SM, CEES | Luciferase activity test | SM: 10–100 μM CEEM: 100–1000 μM | Chinese hamster ovary cells (wild type and nucleotide excision repair (NER) deficient) | NER-competent cells were more resistant to the toxic effects of SM and CEES, indicating the role of NER in repairing DNA damage also in decreasing their toxicity | Matijasevic et al. (2001) |
SM | Quantitative polymerase chain reaction (QPCR) and southern hybridization | SM: 50–500 μM | Human epidermal keratinocytes | SM produced significantly higher levels of both total adducts and crosslink in genomic DNA than mitochondrial DNA DNA inter-strand crosslink introduced as the critical lesion induced by bi-functional alkylating agents | Shahin et al. (2001) |
SM | Agarose gel electrophoresis | SM: 0.01 μM–1000 μM/0–24 h incubation | Human peripheral blood lymphocytes | Exposure to SM caused a time-dependent shift from apoptosis to necrosis (from an oligonucleosome-sized DNA ladder characteristic of apoptotic cell death to a broadband pattern characteristic of necrotic cell death) DNA fragmentation decreased when poly ADP-ribose polymerase (PARP) inhibitors were applied within 8 h of SM exposure | Meier and Millard (1998) |
SM | Gel mobility shift assay | SM: 40–200 μM | Lac UV5 promoter | DNA alkylation by SM preferably occured at; 5′-AA, 5′-GG and 5′-GNC sequences on the DNA | Masta et al. (1996) |
SM | GC-Massa | SM: 131 μM | Human blood (DNA extracted from WBC after exposure) | Identification of 7-(2-hydroxy-ethylthioethyl) guanine as the most abundant adduct, accounted for 61 % of the total alkylation | Ludlum et al. (1994) |
SM | HPLCa analysis of adduct | [35 s] labeled of SM | Human blood and calf thymus DNA | N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine was detected as the abundant adduct | Fidder et al. (1994) |
SM | DNA alkylation and quantitative purine derivatives assay | SM: (0.02–2 mM) | Yeast Saccharomyces cerevisiae | SM induced DNA alkylation independent to cell sensitivity | Kircher and Brendel (1983) |
SM | Giemsa staining of the sites replicating DNA | SM: 0.05 μM/20 min incubation | Primary Syrian hamster fibroblast | The sharp peak of chromatic aberrations was shown 12–16 h after SM-exposure | Savage and Breckon (1981) |
CEES | Ames test | CEES: 0–2 mM | Escherichia coli (repair deficient variants K12, B/r, B) | Identification of mutation sites following CEES; alkylation at the N3 position of adenine and the N7 position of guanine and spontaneous depurination of these alkylated bases Activation of endonuclease II-polymerase I excision-repair system reduced mutagenicity and lethality of CEES | Gilbert et al. (1975) |
SM | Radioactive labeling, X-irradiation, Cytogenetic assay by Orcein staining | SM: 10–1000 ng/ml | Rat lymphosarcoma cell line (Yoshida), Mouse lymphoma cell line (L5178Y)
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