Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin.

A standard operating procedure has been developed for an immunoslotblot assay of sulfur mustard adducts to DNA in human blood and skin for use in a field laboratory. A minimum detectable level of exposure of human blood in vitro (> or = 50 nM) sulfur mustard is feasible with the assay. In the case of human skin, a 1 s exposure to saturated sulfur mustard vapor (830 mg/m(-3)) could still be detected.

An immunochemical assay to detect DNA damage in bovine sperm.

An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of spermatogenesis. A freeze-thaw procedure performed on extended bovine sperm in straws did not induce additional DNA damage immediately after thawing compared with nonfrozen extended sperm. The data suggest that the amount of oxidative damage correlated to the percentage of artificially inseminated cows returning to service within 56 days postinsemination, because a number of sires with high sperm concentrations had a large variation in fertility after artificial insemination. These observations have led to the conclusion that by measuring DNA damage in thawed sperm, one might predict the fertility of bulls with high semen concentration.

Measurement of DNA damage and repair in human white blood cells by an immunochemical assay.

DNA is the most important target molecule for cell killing or the induction of cellular damage by chemical or physical agents. Cell killing depends on the type and amount of damage induced. Exposure to physical or chemical agents can induce a large variety of lesions in DNA: single- and double-stranded breaks, as well as damage to the bases and sugar residues (not leading to a break). It is important to be able to quantify the different types of DNA damage to obtain information about their persistence. The latter may attribute to our understanding of how the various lesions are involved in cell death and/or mutation induction.

(Pheo)melanin photosensitizes UVA-induced DNA damage in cultured human melanocytes.

The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by culturing at two different concentrations, basic (0.01 mM) or high (0.2 mM), of L-tyrosine, the main precursor of melanin. In parallel, pheo- and total melanin contents of the cells were determined. Identical experiments were performed with two melanocyte cultures derived from a skin type I and a skin type VI individual. For the first time the correlation between UVA-induced genotoxicity and pheo-/total melanin content has been investigated. We observed that cultured in basic medium, the skin type VI melanocytes contained 10 times more total melanin and about seven times more pheomelanin than the skin type I melanocytes. Elevation of tyrosine level in the culture medium resulted in an increase of both pheo- and total melanin levels in both melanocyte cultures; however, the melanin composition of skin type I melanocytes became more pheomelanogenic, whereas that of skin type VI melanocytes remained the same. The skin type VI melanocytes cultured in basic medium demonstrated a very high sensitivity (1.18 ssb per 10(10) Da per kJ per m2) toward UVA that is probably related to their high pheo- and total melanin content. Their UVA sensitivity, however, did not change after increasing their melanin content by culturing at high tyrosine concentration. In contrast, the skin type I melanocytes demonstrated a low sensitivity (0.04 ssb per 10(10) Da per kJ per m2) toward UVA when cultured in basic medium, but increasing their melanin content resulted in a 3-fold increase in their UVA sensitivity (0.13 ssb per 10(10) Da per kJ per m2). These results demonstrate that UVA-irradiated cultured human melanocytes are photosensitized by their own synthesized chromophores, most likely pheomelanin and/or melanin intermediates.

Toxicokinetics of sulfur mustard and its DNA-adducts in the hairless guinea pig.

In order to provide a quantitative basis for pretreatment and therapy of intoxications with sulfur mustard (SM) the toxicokinetics of this agent as well as its major DNA-adduct were studied in male hairless guinea pigs for the intravenous, respiratory and percutaneous routes. The study comprised measurement of the concentration-time course of SM in blood and measurement of the concentrations of intact SM and its adduct to guanine in various tissues at several time points after administration of, or exposure to SM. SM was analyzed in blood and tissues by gas chromatography with automated thermodesorption injection and mass-spectrometric detection. DNA-adducts were measured via an immuno-slot-blot method. In contrast with nerve agents of the phosphofluoridate type, SM partitions strongly to various organs, especially the lung, spleen, liver and bone marrow. The respiratory toxicity of SM appears to be local, rather than systemic. Surprisingly, the maximum concentration of SM in blood upon percutaneous exposure to 1 LCt50 (10,000 mg.min.m-3, estimated) is approximately 6-fold higher than that for nose--only exposure to 3 LCt50 (2,400 mg.min.m-3). Pretreatment of hairless guinea pigs with the potential scavengers N-acetyl cysteine or cysteine isopropyl ester did not significantly increase the LCt50-value for nose--only exposure to SM vapor.

Verification of exposure to sulfur mustard in two casualties of the Iran-Iraq conflict.

The exposure of two Iranian victims of the Iran-Iraq conflict (1980-1988) to sulfur mustard was established by immunochemical and mass spectrometric analysis of blood samples taken 22 and 26 days after alleged exposure. One victim suffered from skin injuries compatible with sulfur mustard intoxication but did not have lung injuries; the symptoms of the other victim were only vaguely compatible with sulfur mustard intoxication. Both patients recovered. Immunochemical analysis was based on detection of the N7-guanine adduct of the agent in DNA from lymphocytes and granulocytes, whereas the N-terminal valine adduct in globin was determined by gas chromatography-mass spectrometry after a modified Edman degradation. The valine adduct levels correspond with those found in human blood after in vitro treatment with 0.9 microM sulfur mustard.

Physiological doses of ultraviolet irradiation induce DNA strand breaks in cultured human melanocytes, as detected by means of an immunochemical assay.

An immunochemical assay, i.e. sandwich enzyme-linked immunosorbent assay, has been modified to detect UV-induced damage in cellular DNA of monolayer-grown human melanocytes. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar-simulated light or gamma-rays. Following physiological doses of UVA, UVB or solar-simulated light, a dose-related DNA unwinding comprising a considerable number of single-strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/10(10) Da/kJ/m2 were detected and after UVB irradiation 1.9 ssb/10(10) Da/kJ/m2 were seen. One minimal erythema dose of solar-simulated light induced 2.25 ssb/10(10) Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.

DNA-damage processing in blood lymphocytes of head-and-neck-squamous-cell-carcinoma patients is dependent on tumor site.

It has been reported that an intrinsic susceptibility to cancer is related to the way an individual responds to DNA-damaging agents. The aim of this study was to evaluate whether, in addition to bleomycin-induced chromosomal instability, radiation-induced initial DNA damage and subsequent repair is associated with the development of head-and-neck squamous-cell carcinoma. In this study, 2 assays were performed to measure DNA damage in human peripheral-blood lymphocytes. One was a chromosomal aberration assay which determines sensitivity to chromatid breaks induced by bleomycin, the other an elegant immunochemical assay which measures the level of radiation-induced strand breaks as well as subsequent repair. Age, smoking and alcohol-drinking behavior did not influence the number of chromatid breaks, initial DNA damage or repair capacity. As has been found in previous studies, the mean number of chromatid breaks per cell was significantly different between patients (n = 18) and control persons (n = 19), whereas the amount of initial DNA damage was not. No correlation was found between the outcome of the 2 assays in the subject groups. In contrast to laryngeal-carcinoma patients, oral-cavity-carcinoma patients showed significantly slower repair capacity than controls. Our hypothesis is that the way DNA damage is processed by the patients determines at which site cancer develops in the head and neck area.

A novel type of X-ray-sensitive Chinese hamster cell mutant with radioresistant DNA synthesis and hampered DNA double-strand break repair.

It has been shown that the Chinese hamster cell mutant V-C8 is sensitive to different DNA damaging agents, such as mitomycin C (MMC), alkylating agents, UV light, and X-rays. We found that V-C8 is also sensitive to the following radiomimetic agents: bleomycin (approximately 2-fold, based on D10 values), H2O2 (approximately 2-fold), streptonigrin (approximately 11-fold), and etoposide (approximately 8-fold). Two independent spontaneous MMC-resistant revertants isolated from V-C8 cells show a level of cell killing by X-rays, EMS, and UV light which is similar to that of wild-type cells, suggesting that the observed pattern of cross-sensitivity of V-C8 cells to a wide spectrum of DNA damaging agents results from a single mutation. V-C8 cells also display radioresistant DNA synthesis following gamma-irradiation which, however, remained almost unchanged in the V-C8 revertants. The measurement of the level and rate of repair of DNA single- and double-strand breaks (SSBs and DSBs, respectively) by the DNA elution technique showed that the V-C8 mutant has a slower repair of DSBs induced by gamma-rays. The described unique phenotype of V-C8 cells suggested that V-C8 represents a novel type of mutant amongst X-ray-sensitive hamster cell mutants. To confirm this, complementation analysis with other X-ray-sensitive mutants was performed. V-C8 cells were fused with EM9, XR-1, xrs5, sxi-1, V-3, V-E5, irs3, and BLM2 mutant cells, representing different complementation groups. All the obtained hybrids regained X-ray resistance (or bleomycin resistance in the case of V-C8/BLM2 hybrids) similar to that of wild-type cells, indicating that V-C8 represents a new complementation group. The results presented indicate that V-C8 is defective in a gene involved in a pathway operating in the responses to different DNA damaging agents in mammalian cells.

A modified immunochemical assay for the fast detection of DNA damage in human white blood cells.

An immunochemical assay to detect damage in DNA has been modified to a so-called sandwich ELISA. With this assay DNA damages can be detected that give rise to a certain level of single-strandedness in DNA of white blood cells during partial unwinding of cellular DNA under alkaline conditions. The modified method includes the following steps: incubation of alkali-treated whole blood in the wells of microtiter plates precoated with antibody directed against single-stranded DNA (ssDNA), which results in selective binding of ssDNA, and the subsequent detection of bound ssDNA by incubation with anti-ssDNA antibody alkaline phosphatase conjugate. With this method the amount of damage induced by ionizing radiation in DNA in cells of human blood can be detected within 1 h, after doses as low as 0.2 Gy. The precoating of microtiter plates with anti-ssDNA antibody enables the detection of ssDNA fragments directly in alkali-treated blood samples, isolation of the nucleated cells from the blood is not necessary. Because the DNA is released somewhat faster from lymphocytes than from granulocytes upon alkali treatment, it even appeared possible to discriminate between the effect of the radiation on these cell types in the same blood sample. The method is also applicable to other cell types that can be obtained in suspension.

Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines.

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.

Immunochemical detection of adducts of sulfur mustard to DNA of calf thymus and human white blood cells.

As part of a program to develop methods for dosimetry of exposure to sulfur mustard, we developed immunochemical methods for the detection of the major adduct, N7-[2-[(hydroxyethyl)thio]ethyl]guanine (N7-HETE-Gua), formed after alkylation of DNA with sulfur mustard. After immunization of rabbits with calf thymus DNA treated with sulfur mustard, we obtained the antiserum W7/10 with a high specificity for DNA adducts of sulfur mustard. With this serum, a competitive enzyme-linked immunosorbent assay was developed in which sulfur mustard adducts to DNA could be detected with a minimum detectable amount of 1-5 fmol per well and a selectivity that allows detection of one N7-HETE-Gua among 5 x 10(6) unmodified nucleotides in single-stranded DNA. The complications that arise to isolate double-stranded DNA from biological samples and to make the DNA single-stranded without destruction of the sulfur mustard adducts result in about a 20-fold higher limit for adduct detection in DNA from human blood than in single-stranded DNA. Presently, adducts in white blood cells can be detected after exposure of human blood to sulfur mustard concentrations > or = 2 microM. We synthesized N7-HETE-GMP for use as a hapten to generate monoclonal antibodies against this adduct. After immunization of mice with this adduct coupled to the carrier protein keyhole limpet hemocyanin we obtained several hybridomas producing monoclonal antibodies that recognize N7-HETE-Gua, containing an intact imidazolium ring. The sensitivity of the competitive ELISA with the monoclonal antibodies was comparable to that of the assays performed with the rabbit antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic response and repair of cis-DDP-induced DNA cross-links in the Chinese hamster V79 cell mutant V-H4 which is homologous to Fanconi anemia (group A).

Previously, it has been shown that the V-H4 mutant of Chinese hamster V79 cells is homologous to Fanconi anemia (FA) group A cells. This hamster cell mutant shows a specific sensitivity to DNA cross-linking agents; therefore, the induction and repair of DNA cross-links were studied in V-H4 and wild-type V79 cells after cis-DDP treatment by the DNA alkaline elution technique. A significant difference in repair of these lesions in V-H4 and wild-type cells was observed. After the cis-DDP treatment (24 h) about 3 times more cross-links remained in V-H4 cells in comparison to the parental V79 cells. These results indicate that the process of cross-link repair in V-H4 cells is hampered when compared to that of wild-type cells. To assess the effect of slower removal of DNA cross-links on the mutability of V-H4, the induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase locus (HPRT) by cis-DDP was studied in V-H4 and V79 cells. Despite the increased cytotoxicity of cis-DDP to V-H4 cells, the mutation induction at the HPRT locus was not significantly different in both cell lines, but when the frequency of the hprt mutants was plotted against survival, hypomutability was observed in V-H4 cells after the cis-DDP treatment.

Synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA of calf thymus and human blood.

As part of a program to develop methods for verification of alleged exposure to sulfur mustard, we synthesized and characterized the adducts most likely formed by alkylation of DNA with sulfur mustard: N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine (1), bis[2-(guanin-7-yl)ethyl] sulfide (2), N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine (3), and O6-[2-[(2-hydroxyethyl)thio]ethyl]-guanine and its 2'-deoxyguanosine derivative. Incubation of double-stranded calf thymus DNA and human blood with [35S]sulfur mustard in vitro followed by enzymatic degradation of the DNA and mild depurination afforded three major radioactive peaks upon HPLC analysis. These peaks were identified as 1-3 by coelution with the synthetic markers and mass spectrometric and electronic spectra. Compound 1 appeared to be the most abundant adduct, which is in agreement with previous investigations on DNA alkylation with sulfur mustard.

Quantification of sulfur mustard-induced DNA interstrand cross-links and single-strand breaks in cultured human epidermal keratinocytes.

The induction of DNA interstrand cross-links and their repair has been studied in cultured human epidermal keratinocytes exposed to sulfur mustard, bis-(beta-chloroethyl)sulfide. Alkaline elution is the most sensitive method to determine the number of DNA interstrand cross-links quantitatively. However, in the case of sulfur mustard the reliability of these data will be less since sulfur mustard also induces DNA single-strand breaks (SSB) and/or alkali-labile sites (ALS). The frequency of SSB and/or ALS induced by sulfur mustard is determined immunochemically. Correction for the induction of SSB and/or ALS induced by sulfur mustard resulted in a substantial increase in the calculated number of cross-links. Our results indicate that per microM sulfur mustard approximately 0.05 SSB (and/or ALS)/10(9) Da of DNA and approximately 0.12 cross-links/10(9) Da of DNA were induced immediately after exposure. Most of the DNA interstrand cross-links are removed during the first 24 h post exposure, but a small number of lesions seem to be persistent. In cells exposed to sulfur mustard concentrations as low as 1 microM, repair seems to occur not at all.

Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation.

Alkaline elution has been used for quantitative detection of DNA damage caused by ionizing radiation in unlabeled somatic and germ cells. Both the induction and subsequent repair have been studied for two classes of DNA damage, viz. single-strand breaks (SSB), and base damage (BD) recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of SSB and BD after in vitro exposure of hamster germ cells in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids), and of bone-marrow cells, to biologically relevant doses (0-8 Gy) of 60Co gamma-rays. A dose-dependent increase was observed for both types of lesions, which was similar for most cell types. The elongated spermatids, however, showed a lower induction frequency of SSB (and perhaps BD). Spermatocytes, round spermatids and bone-marrow cells had normal, fast repair of the SSB when compared with the repair reported for cultured rodent cells and human lymphocytes. In contrast, the elongated spermatids showed hardly any SSB repair. The initial rate of repair of BD in spermatocytes and bone-marrow cells was in the same range as that for SSB, but only 60-70% of the initial BD was repaired within 1 h, whereas after that period no SSB were detectable. The round spermatids hardly repaired any BD within the first hour after irradiation, but after 7 h only a few BD could be detected. In elongated spermatids repair of BD could not be measured due to a high background level of this type of damage.

A fourth complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DNA double-strand break repair.

The XR-V9B mutant of Chinese hamster V79 cells which exhibits hypersensitivity to ionizing radiation was isolated by the replica plating technique. The increased sensitivity of XR-V9B cells to X rays (approximately 4-fold, as judged by the D10) was accompanied by increased sensitivity to other DNA-damaging agents such as bleomycin (approximately 17-fold), VP16 (approximately 6-fold), and adriamycin (approximately 5-fold). Only a slightly increased sensitivity was observed after exposure to UV radiation, MMS, or mitomycin C (1.4-, 1.7-, and 2-fold, respectively). As measured by neutral elution after exposure to X rays, XR-V9B cells showed a defect in the rejoining of double-strand breaks (DSBs); after 4 h of repair more than 50% of DSBs remained in comparison to 5% in wild-type cells. No difference was observed in the kinetics of single-strand break rejoining between XR-V9B and wild-type cells, as measured by alkaline elution. To determine whether XR-V9B represents a new complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DSB repair, XR-V9B cells were fused with XR-V15B, XR-1, and V-3 cells, which have impaired DSB rejoining and belong to three different complementation groups. In all cases, the derived hybrids regained the sensitivity of wild-type cells when exposed to X rays, indicating that the XR-V9B mutant represents a new fourth complementation group among X-ray-sensitive Chinese hamster cell mutants defective in DSB repair.

Detection of single-strand breaks and base damage in DNA of blood cells from leukaemia patients receiving chemo- and radiotherapy.

Chemotherapy combined with total-body irradiation (TBI), a conditioning regimen for bone-marrow transplantation (BMT), causes lesions in the cellular DNA of the patients treated. To understand possible consequences of the DNA damage induced during such treatment, information is required about the nature of the damage, the level of induction and its persistence, and about the importance of the various lesions for cell-lethality and/or mutation induction. Recently, we developed a sensitive immunochemical method to quantify single-strand breaks (SSB) in the DNA of mammalian cells. In addition, a modification of the so-called alkaline elution technique was introduced which allows quantification of SSB together with base damage (SSB+BD). These methods have now been applied successfully to study the in vivo induction and repair of DNA damage in WBC of leukaemia patients who prior to BMT were treated with cyclophosphamide (CY) and received TBI. SSB and SSB+BD were determined after two treatments with CY (60 mg kg-1) followed by TBI (4.5-8.6Gy). The CY treatments gave rise to rather persistent SSB. In addition to these, radiation-induced SSB and SSB+BD could be detected shortly after TBI. However, 105 min after TBI, these SSB could be observed no longer, as a result of rapid repair.

A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges.

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

Quantitative detection of DNA damage in cells after exposure to ionizing radiation by means of an improved immunochemical assay.

A simple, sensitive and fast immunochemical method has been developed to quantify the amount of DNA damage in cells of human blood after in vitro exposure to ionizing radiation. The technique is based on the enhancement of the radiation-induced single-strandedness, which occurs in DNA regions flanking strand breaks, by a controlled further unwinding of the DNA in an alkaline solution. Subsequently, the DNA is attached to the wall of polystryene cups by passive adsorption. DNA damage is then quantified by determining the extent of single-strandedness with a monoclonal antibody, D1B, directed against single-stranded DNA. D1B binding is assayed with a 'second' antibody, labelled with either an enzyme or europium. The latter gives slightly more reproducible results. No radioactive labelling of DNA is required and the assay takes only 3.5 h after the collection of blood. Damage can be detected after doses as low as 0.5 Gy. The potential broader application of the method is discussed.